这项工作得到了国家科学基金会的支持（NSF向DB，CMMI-1300920，DB和S.O&#39;M。，CMMI-1531789授予S.O&#39;M。，DB和EAK）和a Busch生物医学研究资助EAK和S.O&#39;M。

聚焦纳秒激光和测量流淌<ol><li> 通过在50 mL玻璃烧杯内放置磁力搅拌棒和多孔消融台来组装消融设备。 注意:消融阶段由3.81厘米直径，1.6毫米厚的不锈钢平台组成，具有十个0.65厘米直径的孔和六个0.50厘米直径的孔，以图1所示的图案钻出。这些孔的目的是允许液体移动穿过靶，使得颗粒不会立即在靶上方积聚。混合不充分导致激光与已经形成的颗粒之间的有害的相互作用。另外，三个＃29（8-32）螺纹孔位于平台周边附近，以接受用作腿的固定螺钉，以提升平台并为磁力搅拌棒提供空间（ 图1A ）。 <ol><li>将烧杯放在磁性上搅拌板并将搅拌板置于xy平移台上，以使得在烧蚀过程中靶材能够移动（ 图1A ）。 </li></ol></li><li>将Nd:YAG激光器设置为基波长为1,064 nm，脉冲持续时间为5 ns，脉冲重复频率为10 Hz。用激光功率和能量计测量每脉冲的能量。所需能量为240-250 mJ。 </li><li>使用250mm焦距会聚透镜（NA = 0.05）将光束聚焦在消融台上的目标下方。 注意:输入光束的半径为4.025 mm，透镜高度为161 mm，可获得所需的光斑尺寸。经验确定最佳斑点大小。利用较大的光斑尺寸来减少溶液中存在的NPs的屏蔽效果。这是平衡的事实，增加斑点大小需要更高的能量以保持足够的能量密度。 </li><li>通过放置金属靶来确定光斑尺寸（参见章节n 2）在舞台上并用几个激光脉冲进行消融。在装有CCD照相机的光学显微镜（4X物镜）上，用千分表滑块查看烧蚀目标以测量斑点尺寸（ 图1A ）。 注意:对于这里的设备，消融系统产生平均面积为5.51平方毫米的斑点大小。每次消融的斑点大小保持在此范围内。 </li><li>通过将脉冲能量除以斑点面积来计算注量。对于这里的设备，注量为4.80J / cm 2 。 </li></ol> 2.通过脉冲激光消融在液体中合成银纳米颗粒<ol><li>使用微量天平称重平面银靶，以获得预消融块。 </li><li>使用双面碳带将银目标粘附到多孔层。向烧杯中加入40 mL消融液（ 图1A ）。目标上方的液体高度为11 mm。 注意:典型的消融液液体是含有60mM SDS或2mM PVP的水溶液以增强单分散性。 </li><li>在持续搅拌下，以0.42厘米/秒的速度将计算机控制的电动xy阶段以0.42厘米/秒的速度移动成椭圆形图案（尺寸:长轴= 2.09厘米，短轴= 0.956厘米，面积= 1.57厘米2 ），并烧制靶20-40分钟。 注意:NPs的浓度随着消融时间的延长而增加。确保搅拌充分活力，以保持NP浓度在整个溶液中均匀，以尽量减少屏蔽效果7 。 </li></ol>表征金属纳米粒子<ol><li>通过倾析从烧杯收集纳米颗粒溶液。通过测量其紫外 - 可见光光谱（200-1100nm）来确认纳米颗粒的存在。 注意:NPs在表面等离子体共振（SPR）波长处具有峰值吸收。对于银，SPR以400nm为中心。高度集中的NP在测量UV-VIS光谱之前，溶液需要稀释，以确保吸光度读数保持在分光光度计的线性范围内。 </li><li> 通过动态光散射（DLS）利用数字分布分析法测量NPs的流体动力学直径29 。 <ol><li>在消融溶液中稀释NP溶液1:40，并将1mL移液到1cm塑料比色皿中。使用180°的测量角度，测量波长为633nm的光散射，以根据斯托克斯 - 爱因斯坦方程确定NP直径: <img alt="公式1" src="/files/ftp_upload/55416/55416eq1.jpg" /> 其中d是流体动力学半径，k是玻尔兹曼常数，T是绝对温度，η是粘度，D是布朗运动的平移扩散系数或速度。 </li></ol></li><li> 使用透射电子微粒确认NP尺寸和形状scopy（TEM） 30 。 注意:使用DLS测量的流体动力学直径大于由于围绕NP的溶剂层使用TEM测量的尺寸。 <ol><li>在双蒸水中稀释NP溶液1:40以除去可能干扰成像的任何过量的添加剂（ 例如 SDS或PVP）。将2μL溶液滴入预先涂有花边/薄碳膜（市售的参见材料清单）的铜TEM网格上，并在室温下在干燥器中真空干燥过夜。 </li><li>对NP进行成像，以评估参考文献30所述的尺寸和形状。 </li></ol></li><li> 为了计算NP浓度，通过将靶放置在含有蒸馏水的超声波水浴中1分钟，从烧蚀的金属靶上移除任何松散附着的NP（步骤2.3）。 <ol><li>在压缩空气流下将目标物干燥1分钟。测量ta的质量在微量天平上。将溶液中的NP的质量定量为消融前后的体重差异，这被认为是将金属纳米粒子喷射到溶液中的结果。 </li></ol></li></ol>后照射<ol><li>在2.2中使用的相同消融溶液中将NPs稀释至最大浓度为100μg/ mL。该浓度限制对于确保均匀照射至关重要。 </li><li>将15-17 mL稀释的NPs转移到含有搅拌棒的石英比色杯中（ 图1B ）。将试管放在与进入的激光平行排列的磁力搅拌板上。 </li><li>使用Nd:YAG激光系统产生25 ps 532 nm激光脉冲和75 mm焦距镜头，将激光对焦于溶液的中心。根据所需的尺寸将溶液照射30分钟至多个小时。 注意:所输送的总能量取决于溶液的浓度和t照射的ime可以在0.5mJ到3.5mJ的范围内。对于这里的装置，透明低浓度样品（&lt;50μg/ mL）的后照射30分钟产生直径为10nm的银NP。 </li></ol> 5.测量纳米颗粒的抗菌性能注意:测试银NPs对革兰氏阳性（ 枯草芽孢杆菌 ）和革兰氏阴性（ 大肠杆菌 ）的毒性 31 。该方法易于适应任何物种;然而，有效剂量的纳米颗粒可能变化很大，必须根据经验确定。这里使用大肠杆菌作为描述该方法的模型系统。 <ol><li>在含有10g / L细菌胰蛋白胨，5g / L酵母提取物和10g / L氯化钠的Luria Broth（LB）中于37℃培养大肠杆菌培养物（菌株MG1655）过夜。将过夜培养物稀释至光密度（λ= 600nm）在新鲜LB中为0.01。 </li><li>如果NPs在含有添加剂（ 如 SDS或PVP）的消融介质中合成，则将相应的化学品加入到LB中，使得浓度在添加NP时保持恒定。 注意:例如，在典型的实验中，将银靶烧成60mM SDS溶液，得到100μg/ mL NPs溶液。如果培养基中NPs的最终浓度为10μg/ mL，则制备含有6mM SDS的LB（ 即在消融液中的1/10浓度的SDS）。当使用这些浓度时，对细菌的生长没有负面影响。这在图3中的-AgNP控制中显示。 </li><li>将NPs添加到稀释的培养物中，浓度范围为0-50μg/ mL，并在37℃振荡培养培养物2小时。作为毒性的阳性对照，用抗生素处理大肠杆菌 （ 例如 30μg/ mL卡那姆）霉素）。 </li><li>孵育2小时后，将培养物样品1:10连续稀释成新鲜的LB，并将每个稀释液的10μL滴定于LB琼脂平板上。通常，10 4 -10 8倍稀释度足以看到单个菌落。 </li><li>一旦液滴被吸收，培养板在37℃过夜，第二天早晨计数菌落形成单位（cfu）。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+size+determination+using+TEM:+a+discussion+of+image+acquisition+and+analysis+for+the+novice+microscopist.">Particle size determination using TEM: a discussion of image acquisition and analysis for the novice microscopist.</a> Langmuir. 24 (20), 11350-11360 (2008).</li><li>Ratti, 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=Irradiation+with+visible+light+enhances+the+antibacterial+toxicity+of+silver+nanoparticles+produced+by+laser+ablation.">Irradiation with visible light enhances the antibacterial toxicity of silver nanoparticles produced by laser ablation.</a> Appl Phys A. 122 (4), 1-7 (2016).</li><li>Sajti, C. L., Sattari, R., Chichkov, B. N., Barcikowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gram+Scale+Synthesis+of+Pure+Ceramic+Nanoparticles+by+Laser+Ablation+in+Liquid.">Gram Scale Synthesis of Pure Ceramic Nanoparticles by Laser Ablation in Liquid.</a> J Phys Chem C. 114 (6), 2421-2427 (2010).</li><li>Streubel, R., Barcikowski, S., Gokce, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+multigram+nanoparticle+synthesis+by+high-power,+high-repetition-rate+ultrafast+laser+ablation+in+liquids.">Continuous multigram nanoparticle synthesis by high-power, high-repetition-rate ultrafast laser ablation in liquids.</a> Opt Lett. 41 (7), 1486-1489 (2016).</li><li>Streubel, R., Bendt, G., Gokce, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilot-scale+synthesis+of+metal+nanoparticles+by+high-speed+pulsed+laser+ablation+in+liquids.">Pilot-scale synthesis of metal nanoparticles by high-speed pulsed laser ablation in liquids.</a> Nanotechnology. 27 (20), 205602 (2016).</li></ol>

作者没有什么可以披露的。

NPs可重现的抗微生物作用需要生产具有相似尺寸和浓度的NPs。因此，标准化激光参数（包括注量，波长和脉冲持续时间）至关重要。虽然动态光散射是估算NP尺寸的一种简单而快速的方法，但尺寸分布的准确量化需要通过TEM直接测量。由于每个激光束在模式分布和散度方面具有不同的特征，因此使用经验过程产生目标上的最佳注量和液体高度是至关重要的。由于可以通过后照射来调节纳米粒子的尺寸，所以可以通过目标质量损失和光密度测量来优化粒子生产的效率。 与常规化学合成方法相比，液体中的激光烧蚀具有在纯水或表面活性剂溶胶中生产纳米颗粒的优点<html> ution。没有前体化合物污染培养物并作为干扰物。研究人员可以在实验室内在短时间内生产纳米颗粒，以便立即使用。该技术高度可重复，不需要专门的培训或人员。然而，重要的是注意这种方法的局限性。首先，通过液体中的激光烧蚀产生的纳米颗粒的形状多样性可以在很宽的范围内变化。如果需要针对特定​​的长宽比或其他形状参数，如果可能，则需要高度迭代的处理。也许这种技术的最大限制是，需要大量纳米粒子的实验将是困难的。可能会出现格氏规模合成，但具有挑战性，需要专门的激光设备32,33,34 。 重要的是注意到许多金属NPs是光敏感的，实际上，用可见光照射银纳米颗粒导致抗菌毒性增加31。增强的功效是由于NP离子释放的增加，因此，重要的是要考虑是否执行PLAL方法并存储保护光束的所得NP。 最后，研究NPs的抗菌效力时，选择表面活性剂和体积排除物（ 如 SDS和PVP）可以降低NP的大小。执行控制实验以确保添加剂本身不具有毒性是重要的。例如， 大肠杆菌以高达10mM的浓度耐受SDS;然而， 枯草芽孢杆菌更敏感31 。因此，当使用枯草芽孢杆菌时，可以将无毒浓度的PVP（2mM）加入到消融液中以获得25nm粒子。 液体中的激光烧蚀以及后照射可用于产生具有一定范围的分散性和尺寸的纳米颗粒分布。这将有助于研究不同的细菌，金属甚至合金。使用PLAL进行纳米颗粒合成提供了一种开发抗菌NPs的新方法，以应对抗菌抗药性日益增长的挑战。

纳米颗粒（NP）通常被定义为具有至少一个尺寸小于100nm长度的颗粒。传统的化学NP合成方法通常需要危险的还原剂，如硼氢化物和肼。相比之下，浸没在液体介质（液体中的脉冲激光消融 - PLAL）中的固体金属靶的激光烧蚀为NP合成提供了一个满足绿色化学1,2的原则的所有12条的环保途径。在PLAL中，通过重复的激光脉冲照射浸没的金属靶。当激光烧蚀靶时，原子簇和蒸汽的致密羽流被释放到其中NPs快速聚结的液体介质中。由PLAL生产的NP细分散在水性介质中，NPs的尺寸，多分散性和组成可以通过改变水性消融液以及激光标准ameters 1，2，3，4，5，6。 可以通过调整激光参数的数量来调整纳米粒子特性，包括:注量，波长和脉冲持续时间（参考文献7 ）。激光能量密度计算为脉冲能量除以目标表面上激光点的面积。能量密度对NPs大小和多分散性的精确影响有些争议。一般来说，已经表明，对于"长"和"超短"脉冲激光系统，存在低和高注射制度，分别产生负面和积极的趋势，分别为8,9,10,11。 NP尺寸分布可以使用诸如动态光散射和透射电子显微镜（TEM）之类的技术来经验性地测量。 激光波长的选择可以影响形成NP的物理机制。在较短的（紫外）波长下，高能光子能够断裂原子间键12 。这种光消融机制是自顶向下的NP合成的一个例子，因为它导致材料的超小碎片的释放，其倾向于在浸没液12,13,14中淬火时产生更大的多分散样品。相比之下，近红外消融（λ= 1,064nm）产生了由等离子体消融12主导的自下而上的合成机制。目标的激光吸收释放与电子碰撞并随后游离的电子。如c部分增加，材料被电离，从而点燃等离子体。周围液体限制等离子体，增强其稳定性，进一步增加吸收12 。当膨胀的等离子体被限制液体淬火时，NPs以各种几何形状4,12,15缩合。 激光脉冲持续时间的选择可进一步影响NP形成过程。常用的长脉冲激光器，脉冲持续时间大于几皮秒，包括所有毫，微，纳米和一些皮秒脉冲激光。在该脉冲宽度方案中，激光脉冲持续时间比电子 - 声子平衡时间长，通常在几皮秒4,16,17,18,19的数量级。这导致能量泄漏到周围的消融介质中，并通过诸如热离子发射，蒸发，沸腾和熔化的热机制形成NPs。 NPs的抗菌活性受粒径21,22,23,24的强烈影响。为了增强尺寸减小和单分散性，可以使用接近NP的表面等离子体共振（SPR）的波长的激光第二次照射NP。入射激光辐射被NP通过激发SPR吸收。 NP的碎裂可能通过热蒸发25,26或库仑爆炸27,28发生 。光激发提高了他的NP温度高于熔点，导致颗粒外层脱落。已经表明，向溶液中加入聚乙烯吡咯烷酮（PVP）或十二烷基硫酸钠（SDS）等添加剂可以大大提高后照射效果5 。在几个报告1,4,6中已经描述了添加各种溶质的影响。通过PLAL易于操作NP特性提供了开发新型NP型抗微生物剂的新方法。

<table><tbody><tr><td>Nanosecond Nd:YAG laser</td><td>Ekspla</td><td>NL303</td><td></td></tr><tr><td>Motorized xy scanning stage</td><td>Standa</td><td>8MTF</td><td></td></tr><tr><td>UV-VIS spectrophotometer</td><td>Agilent</td><td>Cary 60</td><td></td></tr><tr><td>Dynamic light scattering unit</td><td>Malvern</td><td>Zetasizer ZS 90</td><td></td></tr><tr><td>Microbalance</td><td>Maktek</td><td>TM 400</td><td></td></tr><tr><td>Transmission electron microscope</td><td>Zeiss</td><td>EM 902</td><td></td></tr><tr><td>Silver foil target</td><td>Alfa Aesar</td><td>12127</td><td></td></tr><tr><td>250 mm focal length lens</td><td>Edmund Optics</td><td>69-624</td><td></td></tr><tr><td>Copper TEM grids</td><td>Pacific Grid-Tech</td><td>Cu-400LD</td><td>Lacey/thin film coated grid</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; MG1655</td><td>ATCC</td><td>47076</td><td></td></tr><tr><td>Bacto tryptone</td><td>BD Biosciences</td><td>211705</td><td></td></tr><tr><td>Yeast extract</td><td>BD Biosciences</td><td>212750</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP3581</td><td></td></tr><tr><td>Bacto agar</td><td>BD Biosciences</td><td>214010</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Fisher Scientific</td><td>BP166-100</td><td></td></tr><tr><td>Polyvinylpyrrolidone</td><td>Fisher Scientific</td><td>BP431-100</td><td></td></tr><tr><td>Stainless steel disc (for ablation stage)</td><td>Metal Remnants, Inc.</td><td>N/A</td><td>1.5 inch diameter, 16 gauge</td></tr><tr><td>Beaker</td><td>Fisher Scientific</td><td>02-540G</td><td></td></tr><tr><td>Magnetic stir bar</td><td>Fisher Scientific</td><td>14-513-57</td><td></td></tr><tr><td>Magnetic stir plate</td><td>Fisher Scientific</td><td>11-100-49SH</td><td></td></tr><tr><td>Laser energy and power meter</td><td>Coherent</td><td>1098579</td><td></td></tr><tr><td>Carbon tape</td><td>Shinto Chemitron Co. Ltd.</td><td>STR Tape</td><td></td></tr><tr><td>Sonicating water bath</td><td>Branson</td><td>1510</td><td></td></tr><tr><td>Air compressor</td><td>GMC</td><td>Syclone 3010</td><td>For drying ablation target</td></tr><tr><td>75 mm focal length lens</td><td>Edmund Optics</td><td>34-096</td><td>Focusing lens for post-irradiation</td></tr><tr><td>Quartz cuvette</td><td>Precision Cells Inc</td><td>21UV40</td><td>50 mm light path (for post-irradiation)</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>BP906-5</td><td></td></tr><tr><td>Light microscope</td><td>Nikon</td><td>50i</td><td>This microscope is used to focus the laser on the ablation stage. This particular model is no longer available, but any light microscope with a 4X objective will work.</td></tr><tr><td>CCD camera</td><td>AmScope</td><td>MT5000-CCD</td><td></td></tr><tr><td>Micrometer slide</td><td>Ted Pella</td><td>2280-70</td><td></td></tr></tbody></table>

production of metal nanoparticles by pulsed laser-ablation in liquids: a tool for studying the antibacterial properties of nanoparticles

多药耐药细菌的出现是一个全球性的临床问题，导致一些人推测我们恢复了"抗生素"的药物时代。除了确定新型小分子抗微生物药物的努力之外，由于其抗微生物性质，使用金属纳米颗粒作为医疗器械，伤口敷料和消费者包装的涂层也引起关注。可用于纳米颗粒合成的多种方法导致广谱的化学和物理性质，其可以影响抗菌功效。该手稿描述了液体中的脉冲激光消融（PLAL）方法来产生纳米颗粒。该方法允许使用后照射方法以及加入表面活性剂或体积排除物来微调纳米颗粒尺寸，组成和稳定性。通过控制粒径和组成，金属纳米粒子的物理化学性质很大可以探索其可能有助于其抗微生物效力，从而为抗菌发展开辟新的途径。

使用银靶，上述激光参数和消融液中的60mM SDS，产生具有在SPR下的特征UV-VIS吸光度的银NP（ 图2A ）。 TEM和DLS测量显示在照射后约25nm的平均NP直径（ 图2B ）。银靶消融30分钟通常产生200μg/ mL的NP浓度。在评估银NPs的抗菌毒性时，15μg/ mL强烈抑制大肠杆菌生长（ 图3 ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55416/55416fig1.jpg" /> 图1 :设备配置。 （ A ）对于PLAL工艺，在1,064nm波长下工作的Nd:YAG激光器是通过250mm焦距透镜聚焦，以在目标台上产生5.51mm 2的光斑尺寸。使用与光学显微镜耦合的CCD照相机捕获光斑尺寸图像。消融靶设置在具有10个0.65cm直径孔和6个0.50cm直径孔的多孔台上。另外还有3个孔用于固定螺丝，用于支撑搅拌棒上方的台阶。 （ B ）对于后照射，Nd:YAG激光输出设置为532nm，并通过75mm焦距透镜聚焦到含有NPs的石英比色皿的中心。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55416/55416fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55416/55416fig2.jpg" /> 图2 :银纳米粒子的表征。 </s（ A ）银NPs的紫外 - 可见光谱在SPR波长（400nm）处显示特征峰。 （ B ）通过TEM测定后照射后的银NP的尺寸分布。插图显示了AgNP的代表性TEM图像（85,000倍放大倍数，比例尺=100μm）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55416/55416fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55416/55416fig3.jpg" /> 图3 。银纳米粒子的抗微生物作用。用不同浓度的银NP处理大肠杆菌细胞2小时。将连续稀释的培养物铺在LB-琼脂上以测定细菌活力。用30μg/ mL卡那霉素作为阳性对照处理细胞。注意细胞在6mM SDS的存在下，不接受AgNP（-AgNP样品）生长，以确保表面活性剂不独立地导致毒性。该图是来自相同实验的两个板上的菌落的复合物，并且是代表性的结果（n = 5）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55416/55416fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles at JoVE.com

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

金属如铜和银的抗微生物性已经被认识了几个世纪。该协议描述了液体中的脉冲激光消融，一种合成金属纳米粒子的方法，其提供微调这些纳米颗粒的性能以优化其抗微生物效果的能力。

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 ...

production-metal-nanoparticles-pulsed-laser-ablation-liquids-tool-for

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

15.0K 次观看.  Rutgers University-Camden. 金属如铜和银的抗微生物性已经被认识了几个世纪。该协议描述了液体中的脉冲激光消融，一种合成金属纳米粒子的方法，其提供微调这些纳米颗粒的性能以优化其抗微生物效果的能力。 

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

A Method to Fabricate Disconnected Silver Nanostructures in 3D

The standard nanofabrication toolkit includes techniques primarily aimed at creating 2D patterns in dielectric media. Creating metal patterns on a submicron scale requires a combination of nanofabrication tools and several material processing steps. For example, steps to create planar metal structures using ultraviolet photolithography and electron-beam lithography can include sample exposure, sample development, metal deposition, and metal liftoff. To create 3D metal structures, the sequence is repeated multiple times. The complexity and difficulty of stacking and aligning multiple layers limits practical implementations of 3D metal structuring using standard nanofabrication tools. Femtosecond-laser direct-writing has emerged as a pre-eminent technique for 3D nanofabrication.1,2 Femtosecond lasers are frequently used to create 3D patterns in polymers and glasses.3-7 However, 3D metal direct-writing remains a challenge. Here, we describe a method to fabricate silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm. The method enables the fabrication of patterns not feasible using other techniques, such as 3D arrays of disconnected silver voxels.8 Disconnected 3D metal patterns are useful for metamaterials where unit cells are not in contact with each other,9 such as coupled metal dot10,11or coupled metal rod12,13 resonators. Potential applications include negative index metamaterials, invisibility cloaks, and perfect lenses.
In femtosecond-laser direct-writing, the laser wavelength is chosen such that photons are not linearly absorbed in the target medium. When the laser pulse duration is compressed to the femtosecond time scale and the radiation is tightly focused inside the target, the extremely high intensity induces nonlinear absorption. Multiple photons are absorbed simultaneously to cause electronic transitions that lead to material modification within the focused region. Using this approach, one can form structures in the bulk of a material rather than on its surface.
Most work on 3D direct metal writing has focused on creating self-supported metal structures.14-16 The method described here yields sub-micrometer silver structures that do not need to be self-supported because they are embedded inside a matrix. A doped polymer matrix is prepared using a mixture of silver nitrate (AgNO3), polyvinylpyrrolidone (PVP) and water (H2O). Samples are then patterned by irradiation with an 11-MHz femtosecond laser producing 50-fs pulses. During irradiation, photoreduction of silver ions is induced through nonlinear absorption, creating an aggregate of silver nanoparticles in the focal region. Using this approach we create silver patterns embedded in a doped PVP matrix. Adding 3D translation of the sample extends the patterning to three dimensions.

Femtosecond-laser direct-writing is frequently used to create three-dimensional (3D) patterns in polymers and glasses. However, patterning metals in 3D remains a challenge. We describe a method for fabricating silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm.

一个断开连接的银纳米结构的方法来制作的3D

The standard nanofabrication toolkit includes  techniques primarily aimed at creating 2D patterns in dielectric media. Creating  metal patterns on a ...

科研

工程学

Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition

Nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas allows the deposition of metal oxides with tunable morphology, structure, density and stoichiometry by a proper control of the plasma plume expansion dynamics. Such versatility can be exploited to produce nanostructured films from compact and dense to nanoporous characterized by a hierarchical assembly of nano-sized clusters. In particular we describe the detailed methodology to fabricate two types of Al-doped ZnO (AZO) films as transparent electrodes in photovoltaic devices: 1) at low O2 pressure, compact films with electrical conductivity and optical transparency close to the state of the art transparent conducting oxides (TCO) can be deposited at room temperature, to be compatible with thermally sensitive materials such as polymers used in organic photovoltaics (OPVs); 2) highly light scattering hierarchical structures resembling a forest of nano-trees are produced at higher pressures. Such structures show high Haze factor (&gt;80%) and may be exploited to enhance the light trapping capability. The method here described for AZO films can be applied to other metal oxides relevant for technological applications such as TiO2, Al2O3, WO3 and Ag4O4.

We describe the experimental method to deposit nanostructured oxide thin films by nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas. By using this method Al-doped ZnO (AZO) films, from compact to hierarchically structured as nano-tree forests, can be deposited.

制备纳米设计的脉冲激光沉积透明导电氧化物

Nanosecond Pulsed  Laser Deposition (PLD) in the presence of a background gas allows the deposition  of metal oxides with tunable morphology, structure, ...

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) performed on thin sections of rodent tissues: kidneys and tumor, allows the detection of inorganic elements such as (i) Na, Ca, Cu, Mg, P, and Fe, naturally present in the body and (ii) Si and Gd, detected after the injection of gadolinium-based nanoparticles. The animals were euthanized 1&#160;to 24 hr&#160;after intravenous injection of particles. A two-dimensional scan of the sample, performed using a motorized micrometric 3D-stage, allowed the infrared laser beam exploring the surface with a lateral resolution less than 100 &#956;m. Quantitative chemical images of Gd element inside the organ were obtained with sub-mM sensitivity. LIBS offers a simple and robust method to study the distribution of inorganic materials without any specific labeling. Moreover, the compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of the same biological tissue with different types of response:&#160;elemental, molecular, or cellular.

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 &#956;m and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

激光诱导击穿光谱：对于纳米粒子的映射和定量的器官组织的新方法

Emission spectroscopy of laser-induced plasma was applied to elemental analysis of biological samples. Laser-induced breakdown spectroscopy (LIBS) ...

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

可见光和庆大霉素的协同作用<em&gt;假单胞菌假单胞菌</em&gt;微生物

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

免疫与感染

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

生物工程

Studying the Effects of Temperature on the Nucleation and Growth of Nanoparticles by Liquid-Cell Transmission Electron Microscopy

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron microscopy. In this paper, we describe how to prepare an in situ heating experiment for studying the effect of temperature on the formation of gold nanoparticles driven by radiolysis in water. The protocol of the experiment is fairly simple involving a special liquid cell with uniform heating capabilities up to 100 &#176;C, a liquid-cell TEM holder with flow capabilities and an integrated interface for controlling the temperature. We show that the nucleation and growth mechanisms of gold nanoparticles are drastically impacted by the temperature in liquid cell. Using STEM imaging and nanodiffraction, the evolution of the density, size, shape and atomic structure of the growing nanoparticles are revealed in real time. Automated image processing algorithms are exploited to extract useful quantitative data from video sequences, such as the nucleation and growth rates of nanoparticles. This approach provides new inputs for understanding the complex physico-chemical processes at play during the liquid-phase synthesis of nanomaterials.

Temperature control during liquid-phase electron microscopy experiments opens up new perspectives of studying the dynamic of nanoparticles in liquid environments mimicking their formation or application media. Using recently developed heating liquid cells, we directly observed the influence of temperature on the nucleation and growth processes of gold nanoparticles in water.

研究温度对液细胞传输电子显微镜对纳米粒子核化和生长的影响

Temperature control is a recent development that provides an additional degree of freedom to study nanochemistry by liquid cell transmission electron ...

化学

通过激光烧蚀合成纳米颗粒和纳米结构

Ultrafast Laser-Ablated Nanoparticles and Nanostructures for Surface-Enhanced Raman Scattering-Based Sensing Applications

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields such as sensing, catalysis, and medicine. The exceptional feature of this technique is the formation of nanoparticles (colloids) and nanostructures (solids) in a single experiment with ultrashort laser pulses. We have been working on this technique for the past few years, investigating its potential using the surface-enhanced Raman scattering (SERS) technique in hazardous materials sensing applications. Ultrafast laser-ablated substrates (solids and colloids) could detect several analyte molecules at the trace levels/mixture form, including dyes, explosives, pesticides, and biomolecules. Here, we present some of the results achieved using the targets of Ag, Au, Ag-Au, and Si. We have optimized the nanostructures (NSs) and nanoparticles (NPs) obtained (in liquids and air) using different pulse durations, wavelengths, energies, pulse shapes, and writing geometries. Thus, various NSs and NPs were tested for their efficiency in sensing numerous analyte molecules using a simple, portable Raman spectrometer. This methodology, once optimized, paves the way for on-field sensing applications. We discuss the protocols in (a) synthesizing the NPs/NSs via laser ablation, (b) characterization of NPs/NSs, and (c) their utilization in the SERS-based sensing studies.

作者聚焦:通过液体中的激光烧蚀进行纳米颗粒合成的进展和应用 

Ultrafast laser ablation in liquid is a precise and versatile technique for synthesizing nanomaterials (nanoparticles [NPs] and nanostructures [NSs]) in liquid/air environments. The laser-ablated nanomaterials can be functionalized with Raman-active molecules to enhance the Raman signal of analytes placed on or near the NSs/NPs.

超快激光烧蚀纳米颗粒和纳米结构在基于表面增强拉曼散射的传感应用中的应用

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

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

摘要

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超快激光烧蚀纳米颗粒和纳米结构在基于表面增强拉曼散射的传感应用中的应用

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素