Name: preparation and photoacoustic analysis of cellular vehicles containing gold nanorods
Uploaded: 2016-05-02T13:00:00.000+00:00
Duration: 10 min 46 s
Description: Watch this Scientific Journal Video about Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods at JoVE.com

这项工作是部分由托斯卡纳大区和欧洲共同体的ERANET +项目LUS泡沫和BI-TRE的框架内支持。

注:金纳米棒的全部浓度是名义上金摩尔浓度表示。为与其它工作相比较，注意，1M的金大致相当于20μM的金纳米棒，在我们的情况。 1.阳离子金纳米棒的制备注意:该方法开始于西曲溴铵（CTAB）加帽的金纳米棒通过用抗坏血酸自催化还原氯金酸4的合成，根据根据Ratto 等人 26通过Nikoobakht 等 25介绍，并适于协议。然后这些金纳米棒是为了获得更多的生物相容性和亲和性血浆膜，由聚乙二醇的组合改性导线束10，11，27，28和季铵化合物22。 <ol><li>净化24毫升CTAB皑皑的金纳米棒在concentratio通过离心的两个周期（12,000 xg离心，30分钟）和倾析450微米的Au的n个。确保死体积至初始体积的比例大约是1/200或更低在这一协议的所有的离心步骤。使用500μM的水溶液CTAB作为洗涤溶液，并在含有500μM的CTAB和0.005％（体积/体积）聚山梨酯20的pH 5的颗粒最后转移到6毫升的100mM乙酸盐缓冲液。 </li><li>添加30微升10毫摩尔含水的α-甲氧基ω-巯基聚（乙二醇）（MW〜5000），并离开在37℃下30分钟以发生反应。 </li><li>添加30微升100mM（11-Mercaptoundecyl） - N，N，N-三甲基溴化物在二甲基亚砜和静止在37℃下放置24小时。 </li><li>接着，通过离心分离的四个周期（12,000 xg离心，30分钟）和倾析的水加18毫升0.005％（体积/体积）聚山梨醇酯20和纯化这些颗粒。在水使用0.005％（体积/体积）聚山梨醇酯20作为洗涤溶液并在pH颗粒最后转移到2.4毫升无菌PBS7.4。黄金最终标称浓度为4.5毫米。 </li></ol> 2.小​​鼠巨噬细胞的装载与金纳米棒<ol><li>使用单核细胞/巨噬细胞细胞系J774A.1和DMEM补充有10％胎牛血清，1 2mM谷氨酰胺，100单位/ ml青霉素和100μg/ ml链霉素的培养基。板5×10 5个细胞在60mm直径四个培养皿中，并允许他们生长24小时，以便在分离的时间被亚汇合（见步骤2.2）。 <ol><li>整个协议，保持标准培养条件下（37℃，5％CO 2，95％空气和100％相对湿度）下的细胞。使用层流柜和适当的个人防护装备操纵细胞。 </li></ol></li><li> 24小时后，装入阳离子金纳米棒的细胞，并准备他们被嵌入到壳聚糖膜: <ol><li>为了让由细胞吸收的颗粒中，添加4.5等分试样毫凹阳离子金纳米棒在PBS到每个培养皿，从而达到100μM的Au中的最终浓度。留在孵化培养皿24小时。 </li><li>接着，观察在光学显微镜下的细胞以确认其良好的条件和上载。细胞应表现出自己的正常形态和一些深色的细胞内囊泡。用刮刀分离细胞，合并那些来自两个培养皿中，以实现细胞的合适量为以下步骤（至少2×10 6个细胞），并离心它们（120 XG，6分钟），以消除任何多余的阳离子金纳米棒。该细胞团看起来应该几乎是黑色的。 </li><li>暂停于2ml PBS中沉淀并通过使用Bürker室的细胞计数。离心机含有2×10 6个细胞（120 XG，6分钟），并固定其沉淀在2ml 3.6％（重量/体积）在PBS中的甲醛在室温下十分钟悬浮液。最后，通过centrifugat洗此沉淀三次离子（120 XG，6分钟）以除去固定液。用PBS洗涤液。 </li></ol></li></ol> 3.埋入巨噬细胞的成壳聚糖膜注:壳聚糖26，27，28的特殊属性，29用来生产含阳离子金纳米棒染色的巨噬细胞仿生幻影。相对于其它的水凝胶诸如琼脂糖，壳聚糖使薄膜是强得多和更薄，这是用于PA显微镜6的关键。这些体模的制造是按照以前的协议29，30，31与作为规定在以下30一些修改进行。 <ol><li>制备酸化（pH 4.5，通过加入乙酸获得）和粘性3％（重量/体积）的低分子量脱乙酰壳多糖（平均分子量120 kDa）的溶液，充分混合，并让它在40℃均化24小时。 </li><li>接着，混合含阳离子金纳米棒（2×10 6个细胞）用500μl壳聚糖溶液的鼠巨噬细胞。 </li><li>为了获得〜50微米厚的幻影，倒入250毫克混合物的成1.91厘米2聚苯乙烯模具和离开他们在氮气流下24小时。此后，将这些样品与500微升的1M NaOH，以诱导交联，并用10ml超纯水的冲洗。 </li></ol> 4.光声转换的稳定性测试注:PA转化的稳定性由在参考文献6中描述的PA实验用自制的设置装置的影响。 <ol><li>通过使用塑料夹持器浸渍在培养皿的暂停含有在DI水中的巨噬细胞壳聚糖薄膜，例如，以保持从板的底面〜5mm的距离。将这个盘到微米级的XY工作台为了控制样本位置。 </li><li>聚焦用〜5纳秒脉冲持续时间的激光束在共振与金纳米棒（ 例如纵向电浆频带，从通过Q开关Nd的三次谐波泵浦光参量振荡器:YAG激光波长为400 - 2500 5纳秒的纳米和脉冲持续时间）垂直于与〜300微米的光斑直径在膜表面。 <ol><li>放置在激光出射前方的衰减器来调谐激光能量密度，并使用分束器来聚焦激光束的一部分，以能量计（ 例如 ，热电检测器）和监测注量的波动。保持对准期间低于〜光能量密度1兆焦耳/ 平方厘米每脉冲。每当激光是使用合适的激光护目镜。 </li></ol></li><li>浸超声换能器（1频率范围 - 20兆赫）到培养皿〜2毫米离膜表面的，并通过使用测微翻译调节其位置和旋转阶段最大限度地从电影发出的声信号。 </li><li>确定的探针通量楼罗不会损坏样品6: <ol><li>在约1毫焦/厘米2每脉冲，至少500次脉冲的注量照射样品的随机点。对于每一个脉冲，收购超声换能器和激光能量密度从示波器电能表相应的声信号。命名平均能量密度为F LO 的审判 。 </li><li>计算PA的信号作为其峰 - 峰振幅为脉冲数的函数的强度。为了配重的激光强度的波动，正常化每个声信号自身注量至F LO 试验的比率的幅度。分析的归一化的PA的强度作为脉冲数的函数的趋势并验证其随时间的稳定性。 </li><li>在不稳定的情况下，重复步骤4.4.1具有F LO的较低值至4.4.3 <s达&gt;审判。在稳定的情况下，重复使用F LO 试验的值由〜10％以上，直到稳定性的损失就出现了。设置探头通量楼罗为确保稳定性楼罗 审判的第二高值。 </li></ol></li><li>衡量一个重塑的阈值能量密度: <ol><li>选择样本的另一随机点以上以F LO 500的脉冲探测的平均PA强度（I LO 一个 ）。 </li><li>设置名义通量除了F LO更大，并提供50个脉冲。命名他们的平均能量密度为F EXC。 </li><li>再次探测，平均PA的强度（I LO 二 ）在以F LO 500脉冲。计算比R = I LO B / I LO 一个 。下面统一为R的值给出了样品的光学性质的不可逆的变化的证据。 </li><li>使用测微XY工作台移动薄膜，改变POIN随机样本吨。重复步骤4.5.1使用F EXC不同的值4.5.4，以便采取几个值的R的下方，左右及以上的统一。 15分是合理的。 </li><li>情节ř为F EXC的函数，并确定第作为该值的整形阈通量˚F当R开始从一个超越统计不确定性不同。6 </li></ol></li></ol>

作者宣称，他们没有竞争的经济利益。

靶向肿瘤相关巨噬细胞的概念正在成为一个强大的概念，以打击癌症34，35，36。在这里，而不是销毁，这些细胞被招募为移动车辆，使金纳米棒成肿瘤，其取向的剥削。这个角度看，需要颗粒的贴心设计，融入细胞及其鉴定。我们已发现，装载有阳离子金纳米棒的鼠巨噬细胞的耐光不会从颗粒的内吞囊泡内的限制，这意味着他们的电浆耦合和交叉过热不是...

在过去十年中，各种电浆颗粒如金纳米棒，纳米壳和纳米笼，在生物医学光学1，2，3，4已受到相当关注的应用。在与标准的金纳米球方差，这些较新的粒子共振在于提供用于通过主体最深光学穿透并用内源性组分1最高光学对比度的近红外（NIR）窗口。这个特性已引起对创新应用，例如光声（PA）的成像和癌症的光热消融的兴趣。然而，有几个问题限制这些颗粒的临床渗透。例如，它们的光学活性趋于诱导它们过热和修改他们的功能性形状朝向多个球形轮廓，其驱动一个光稳定性5，6，7，8 </SUP&gt; 9。占主导地位的科学辩论的另一个问题是他们的全身输送到肿瘤。尤其是金纳米棒相结合，是理想渗透了该显示增强通透性和保留和易用性恶性肿瘤标志物的特异性探针结合的肿瘤大小。因此，他们对直接注射到血流中制备被认为是一个可行的方案10，11，12，13。然而，这条路线仍然是个问题，其中大部分粒子变得由单核吞噬细胞系统10，11，12捕获。此外，另一个问题是，通过主体14循环后的粒子的光学和生化稳定性。当颗粒失去胶体稳定性和聚集，其等离子的特点和传热动态可能从电浆子耦合15受苦， </s达&gt; 16，17和交叉过热18。 最近，利用肿瘤相关巨噬细胞的趋性的概念已经成为一个聪明的替代19，20，21。这些细胞持有检测和高特异性的肿瘤普遍存在一种与生俱来的能力。因此，一个角度看可能是这些细胞从病人隔离， 与体外金纳米棒装载它们，然后注入回到患者，以意向使用它们作为负责递送的细胞的车辆。另一个优点是获得对颗粒的光学和生化稳定性更多的控制，因为它们的生物界面将在体外构建。尽管如此，这些细胞汽车作为光造影剂的性能需要一个批判性的分析。 在这项工作中，我们描述了准备和cellul的关键问题含有金纳米棒为癌症的PA的成像芳车辆。 PEG化的金纳米棒被修改与季铵化合物22，以实现该预期以促进与血浆膜23,24及其相互作用的阳离子分布。这些颗粒进行有效和非特异性摄取大部分细胞种类，希望不受外界干扰太多与他们的生物学功能。小鼠巨噬细胞装载有高达每细胞多达200，000阳离子金纳米棒，这成为紧内吞囊泡内密闭。这种配置应该出现的问题，因为这些囊泡内电浆耦合和交叉过热的威胁。因此，巨噬细胞被嵌入模仿生物组织生物聚合物水凝胶，以验证大多数颗粒PA转化的稳定性被保持在从生长培养基转移到内吞囊泡。有效实现了È测量标准，以便测量的PA转化为PA的成像直接兴趣的条件下的稳定性制定。一个重塑阈值设置在光不稳定性的非常发病50的激光脉冲10赫兹的典型重复率一个序列后。 我们相信，这些结果可能为新的发展战略，以实现等离子粒子肿瘤提供动力。

<table><tbody><tr><td>Hexadecyltrimethylammonium bromide</td><td>Sigma-Aldrich</td><td>H6269</td><td>To synthesize gold nanorods</td></tr><tr><td>Gold(III) chloride trihydrate</td><td>Sigma-Aldrich</td><td>520918</td><td>To synthesize gold nanorods</td></tr><tr><td>Silver nitrate</td><td>Sigma-Aldrich</td><td>S6506</td><td>To synthesize gold nanorods</td></tr><tr><td>L-ascorbic acid</td><td>Sigma-Aldrich</td><td>A5960</td><td>To synthesize gold nanorods</td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td></td><td>To synthesize gold nanoseeds</td></tr><tr><td>MeO-PEG-SH</td><td>Iris Biotech</td><td>PEG1171</td><td>To PEGylate gold nanorods. Molecular weight about 5,000 Da.</td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>320099</td><td>To PEGylate gold nanorods and solubilize chitosan</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S8750</td><td>To PEGylate gold nanorods</td></tr><tr><td>(11-Mercaptoundecyl)-&lt;em&gt;N,N,N&lt;/em&gt;-trimethylammonium bromide</td><td>Sigma-Aldrich</td><td>733305</td><td>To modify gold nanorods with quaternary ammonium compounds</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>276855</td><td>To solubilize (11-mercaptoundecyl)-&lt;em&gt;N,N,N&lt;/em&gt;-trimethylammonium bromide</td></tr><tr><td>Polysorbate 20</td><td>Sigma-Aldrich</td><td>P2287</td><td>To centrifuge PEGylated gold nanorods</td></tr><tr><td>PBS</td><td>Lonza</td><td>BE17-516F</td><td>To suspend gold nanorods before incubation with cells and to treat pellets of cells</td></tr><tr><td>J774a.1</td><td>ATCC</td><td>TIB-67</td><td>Monocyte/macrophage murine cell line</td></tr><tr><td>DMEM</td><td>Lonza</td><td>BE12-707F</td><td>Cell culture medium</td></tr><tr><td>FBS</td><td>Lonza</td><td>DE14-801F</td><td>To be added to cell culture medium</td></tr><tr><td>L-glutamine</td><td>Lonza</td><td>BE17-605E</td><td>To be added to cell culture medium</td></tr><tr><td>Penicillin/streptomycin</td><td>Lonza</td><td>DE17-602E</td><td>To be added to cell culture medium</td></tr><tr><td>Petri dish</td><td>NEST</td><td>705001</td><td>Cell culture dish</td></tr><tr><td>Cell scraper</td><td>EuroClone</td><td>ES7018</td><td>To detach cells</td></tr><tr><td>Formaldehyde</td><td>Fluka</td><td>47630</td><td>To fix cells</td></tr><tr><td>Chitosan, low molecular weight</td><td>Sigma-Aldrich</td><td>448869</td><td>75-85% deacetylated. Molecular weight about 120,000 Da.</td></tr><tr><td>Sodium hydroxyde</td><td>Sigma-Aldrich</td><td>306576</td><td>To insolubilize chitosan and generate the hydrogel</td></tr><tr><td>Polystyrene cell culture plates</td><td>NEST</td><td>702011</td><td>Used as molds to fabricate chitosan hydrogels</td></tr><tr><td>Optical parametric oscillator pumped by the third harmonic of a Q-switched Nd:YAG laser</td><td>Continuum, Santa Clara, USA</td><td>Surelite OPO plus</td><td>Source of optical excitation for photoacoustic tests</td></tr><tr><td>Pyroelectric detector&amp;nbsp;</td><td>Gentec, Quebec, Canada</td><td>QE8SP</td><td>To monitor optical fluence for photoacoustic tests</td></tr><tr><td>Pre amplified needle hydrophone</td><td>Precision Acoustic, Dorset, UK</td><td>Model with 1 mm sensor diameter and 1-20 MHz frequency range</td><td>To measure photoacoustic signals</td></tr></tbody></table>

preparation and photoacoustic analysis of cellular vehicles containing gold nanorods

金纳米棒是为一系列的生物医学应用，如光热消融和癌症的光声成像的吸引力，这要归功于在近红外窗口，低细胞毒性和潜在的对置成肿瘤其强烈的光学吸光度。然而，他们交付的肿瘤仍然是一个问题。一种创新的方法包括可装在体外金纳米棒肿瘤相关巨噬细胞的向性的开采。在这里，我们描述的制备方法和含有金纳米棒的细胞车辆的光声检查。 PEG化的金纳米棒被修改与季铵化合物，以实现一种阳离子轮廓。与普通培养皿小鼠巨噬细胞的接触，这些粒子被发现发生巨大摄取进入内吞囊泡。然后将这些细胞被包埋在生物聚合物水凝胶，这是用来验证光声转换的稳定性的颗粒被保持在其纳入蜂窝车辆。我们相信，这些结果可能为新型的发展战略提供等离子粒子肿瘤新的灵感。

这里，含有金纳米棒为癌症的PA的成像蜂窝车辆的可行性与协议的典型的结果一起示出。 图1中的TEM图像显示步骤1之后的颗粒和它们的细胞车辆步骤2.颗粒和细胞的TEM成像的制备别处17描述后的通常外观。阳离子金纳米棒进行巨噬细胞中一个巨大的积累，维持其正常形态。被发现的粒子被严密囊泡内吞局限?...

Watch this Scientific Journal Video about Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods at JoVE.com

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

含有金纳米棒蜂窝车辆的制备及光声分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

preparation-photoacoustic-analysis-cellular-vehicles-containing-gold

Research

JoVE Journal

Bioengineering

6.8K 次观看.  Italian National Research Council. We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

视频: Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

科研

生物工程

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

根据环境条件的金纳米粒子的稳定低聚簇的大小控制合成的一种简便方法

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

化学

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

金纳米棒基于对苯二酚合成

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

细胞物质相互作用研究微细的纳米多孔金模式

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Construction and Operation of a Light-driven Gold Nanorod Rotary Motor System

The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and artificial nanomotors and may provide new routes towards single cell analysis, studies of non-equilibrium thermodynamics, and mechanical actuation of nanoscale systems. A facile way to drive rotation is to use focused circularly polarized laser light in optical tweezers. Using this approach, metallic nanoparticles can be operated as highly efficient scattering-driven rotary motors spinning at unprecedented rotation frequencies in water.
In this protocol, we outline the construction and operation of circularly-polarized optical tweezers for nanoparticle rotation and describe the instrumentation needed for recording the Brownian dynamics and Rayleigh scattering of the trapped particle. The rotational motion and the scattering spectra provides independent information on the properties of the nanoparticle and its immediate environment. The experimental platform has proven useful as a nanoscopic gauge of viscosity and local temperature, for tracking morphological changes of nanorods and molecular coatings, and as a transducer and probe of photothermal and thermodynamic processes.

Plasmonic gold nanorods can be trapped in liquids and rotated at kHz frequencies using circularly-polarized optical tweezers. Introducing tools for Brownian dynamics analysis and light scatteringspectroscopy leads to a powerful system for research and application in numerous fields of science.

轻型金纳米旋转电机系统的施工与运行

The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and ...

工程学

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

生物化学

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 ...

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

金纳米棒辅助神经细胞的刺激光

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

神经科学

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

生物应用近红外发光金纳米簇的合成

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

直接合成金纳米颗粒用于蛋白质定位分析

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP).
Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking.
A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

作者聚焦：在细胞中直接合成 EM-Visible 金纳米颗粒，用于具有保存完好的超微结构的蛋白质定位分析  

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.