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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
聚焦纳秒激光和测量流淌 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?…
Representative Results
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 ）。 <p class="jove_content" fo:keep-togeth…
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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'M。，CMMI-1531789授予S.O'M。，DB和EAK）和a Busch生物医学研究资助EAK和S.O'M。
Materials
| Nanosecond Nd:YAG laser | Ekspla | NL303 | |
| Motorized xy scanning stage | Standa | 8MTF | |
| UV-VIS spectrophotometer | Agilent | Cary 60 | |
| Dynamic light scattering unit | Malvern | Zetasizer ZS 90 | |
| Microbalance | Maktek | TM 400 | |
| Transmission electron microscope | Zeiss | EM 902 | |
| Silver foil target | Alfa Aesar | 12127 | |
| 250 mm focal length lens | Edmund Optics | 69-624 | |
| Copper TEM grids | Pacific Grid-Tech | Cu-400LD | Lacey/thin film coated grid |
| E. coli MG1655 | ATCC | 47076 | |
| Bacto-tryptone | BD Biosciences | 211705 | |
| Yeast extract | BD Biosciences | 212750 | |
| Sodium chloride | Fisher Scientific | BP3581 | |
| Bacto-agar | BD Biosciences | 214010 | |
| Sodium dodecyl sulfate | Fisher Scientific | BP166-100 | |
| Polyvinylpyrrolidone | Fisher Scientific | BP431-100 | |
| Stainless steel disc (for ablation stage) | Metal Remnants, Inc. | N/A | 1.5 inch diameter, 16 gauge |
| Beaker | Fisher Scientific | 02-540G | |
| Magnetic stir bar | Fisher Scientific | 14-513-57 | |
| Magnetic stir plate | Fisher Scientific | 11-100-49SH | |
| Laser energy and power meter | Coherent | 1098579 | |
| Carbon tape | Shinto Chemitron Co. Ltd. | STR Tape | |
| Sonicating water bath | Branson | 1510 | |
| Air compressor | GMC | Syclone 3010 | For drying ablation target |
| 75 mm focal length lens | Edmund Optics | 34-096 | Focusing lens for post-irradiation |
| Quartz cuvette | Precision Cells Inc | 21UV40 | 50 mm light path (for post-irradiation) |
| Kanamycin | Fisher Scientific | BP906-5 | |
| Light microscope | Nikon | 50i | 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. |
| CCD camera | AmScope | MT5000-CCD | |
| Micrometer slide | Ted Pella | 2280-70 |
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Citazione di questo articolo
Ratti, M., Naddeo, J. J., Griepenburg, J. C., O’Malley, S. M., Bubb, D. M., Klein, E. A. Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles. J. Vis. Exp. (124), e55416, doi:10.3791/55416 (2017).