Name: synthesis of immunotargeted magneto-plasmonic nanoclusters
Uploaded: 2014-08-22T16:00:00.000+00:00
Duration: 9 min 43 s
Description: Watch this Scientific Journal Video about Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters at JoVE.com

这项工作是由美国国立卫生研究院资助R01 EB008101和R01 CA103830部分支持。

1，仪器及玻璃制品的制备<ol><li>穿戴适当的防护设备， 即一个白大褂，一次性手套和护目镜。 </li><li>圆底烧瓶连接到冷凝器和浸在硅油浴与温度计的温度监测。放的热量下的油浴中（ 图1）的来源（ 例如，热板）。使用能够测量温度高于260℃的温度计。 </li></ol> 2，初级合成的混合磁等离激元纳米粒子<ol><li>使磁芯纳米粒子<ol><li>加入353.2毫克（1毫摩尔），铁（III）乙酰丙酮化物，将1ml（2毫摩尔）油酸，将1ml（2毫摩尔）的油胺，1.292克（5毫摩尔）1,2 - 十六烷和10ml苯醚为圆汉字瓶。 </li><li>大力使用磁力搅拌棒，加热至250-260℃，回流1小时搅拌混合。然后，等待溶液冷却至室温。确保温度低于260℃，以防止苯基醚的沸点和防止突发的反应混合物从圆底烧瓶中，向冷凝器。 注意:反应混合物是非常热和化学物质可能引起刺激。操作必须在通风橱和穿戴适当的个人防护装备。保证充分的通风的油浴。 注:合成的磁性纳米粒子的过程中，油浴中保持在250-260ºC温度1小时。在原则上，派热克斯玻璃皿可以被用于此目的。然而，最大的连续温度为硼硅玻璃是〜260ºC根据供应商的信息。因此，金属容器提供的，因为它可以承受更高的温度，并在多个运行持续时间越长，反应更安全的选择。 </li></ol></li><li>沉积金壳上的磁性纳米粒子核<ol><li>添加411.5毫克（1.1毫摩尔）金王牌的tate，加入0.25ml（0.75毫摩尔）油酸，将1.5ml（3.0毫摩尔）的油胺，775.3毫克（3毫摩尔）1,2 - 十六烷和15ml苯醚至圆底烧瓶中。 </li><li>加入5 mL悬浮磁性纳米颗粒的步骤2.1。将反应混合物加热至180℃并保持在回流下搅拌1小时。等待该溶液冷却至室温。 </li><li>加入50毫升乙醇中，以沉淀混合初级纳米颗粒，然后离心以3250×g的15分钟。 </li><li>用水浴超声悬浮于25ml正己烷沉淀。加25毫升乙醇中，以沉淀出主混合纳米粒子。离心机在3250×g离心15分钟，重悬沉淀在己烷中。重复此步骤三次。 </li><li>干燥在真空干燥器中O 2 / N的沉淀主要混合纳米粒子。确认该颗粒是完全干燥的。 </li></ol></li></ol> 3，混合动力磁等离子团簇合成和大小分离<ol><li&gt;重悬于5毫克干初级混合纳米粒子在1毫升己烷用水浴超声。超声处理的纳米颗粒悬浮液，直到没有可见沉淀物的存在。 </li><li>从步骤加入该溶液3.1至10ml的十二烷基硫酸钠（2.8毫克/毫升）水溶液中以附帽20毫升玻璃小瓶中。添加的主混合纳米颗粒滴悬浮液一滴地避免了两个阶段的下一个步骤之前混合。 </li><li>超声处理在水浴超声处理2小时的两相溶液中，然后通过加热的水浴中，在80℃下进行10分钟。等待该溶液冷却至室温。 <ol><li>填充水的超声浴中的操作电平线。居中在超声处理浴中的玻璃小瓶中。一种乳液形式立即在两相之间。摇通过超声处理的开始之后手的两相溶液;这有利于含伯混合纳米颗粒的相和底部的水相之间的混合。 注:请注意THAT中的超声仪2小时手术后会热起来。 </li></ol></li><li>离心反应混合纳米簇悬浮液在100×g离心30分钟。同时收集沉淀物和上清液。悬浮于0.1 mM柠檬酸钠沉淀物在10分钟超声。纳米团簇的预期大小是直径〜180纳米。 </li><li>步骤3.3将上清转移到一个新的锥形管。 </li><li>离心从步骤3.4的悬浮液在400×g离心30分钟。同时收集沉淀物和上清液。悬浮于0.1 mM柠檬酸钠沉淀物在10分钟超声。纳米团簇的预期大小是直径〜130纳米。 </li><li>步骤3.5将上清转移到一个新的锥形管。 </li><li>离心从步骤3.6的悬浮液在1500×g离心30分钟。收集0.1 mM柠檬酸钠沉淀重悬在10分钟超声。纳米团簇的预期大小是直径约90纳米。 </li><li>加入300 _6;升纳米簇悬浮液至96孔微板读数器，用于测量紫外 - 可见 - 近红外吸收光谱。下降10微升悬浮液中纳米团簇上的碳包覆铜网格TEM成像。 </li></ol> 4，共轭单克隆抗体的制备纳米团簇<ol><li>制备100μl的单克隆抗体溶液（1毫克/毫升）的PBS，pH为7.2， 例如 ，抗表皮生长因子受体2（HER2）的抗体或抗表皮生长因子受体1（EGFR）抗体。 </li><li>步骤4.1抗体溶液中加入3.9毫升4毫米肝素钠，pH值7.2。离心机通过10 K截留分子量离心过滤的解决方案，在3,250×g离心20分钟，在8℃。重悬的抗体在4毫米的HEPES，pH值7.2，于100微升的最终体积。 注意:此步骤是，以取代在与HEPES的抗体溶液的原始媒体。 </li><li>加入10微升100mM的NaIO 4至100μl的抗体溶液。覆盖所述反应小瓶用一个人的使用轨道摇床uminum箔在RT，搅拌30分钟。 </li><li>淬灭，加入500μl的1×PBS中的反应。 </li><li>加入2微升46.5毫米的连接器解决方案（dithiolaromatic PEG6-CONHNH 2）抗体溶液从步骤4.4振摇1小时在室温。 </li><li>过滤器采用10 K截留分子量离心过滤器的解决方案，在3,250×g离心20分钟，在8℃。重新悬浮于1×PBS中的抗体至100μl导致的〜1毫克/毫升抗体浓度的最终体积。 </li><li>从步骤4.6（1毫克/毫升）120分钟在室温下混合100微升纳米簇悬浮在OD〜1.0用1μl的修饰抗体。 </li><li>加入10微升10 -3 M 5 kDa的巯基聚乙二醇，振摇15分钟在室温。 </li><li>离心反应溶液在830×g下3分钟。弃上清，重悬在100微升2％的泥沙w / v的5 kDa的PEG在PBS，pH值为7.2。 </li><li>测定抗体偶联的纳米团簇的吸收光谱，并比较吨他吸光度裸露的纳米团簇的光谱。期待一对夫妇的结合后，纳米红移。 </li><li>如果纳米粒子团聚物由显著移与在红色-近红外区域的增加OD所示，增加硫醇的PEG的浓度为5×10 -3 M。此外，增加了孵育时间与巯基的PEG至30分钟，并降低了离心速度在200 XG增量。 </li><li>对于癌症细胞标记测试中，从步骤4.9到癌症细胞悬浮液在中等或1×PBS中（1毫升〜10 6个细胞）中添加的抗体缀合的纳米颗粒，并混合60分钟。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

在成功地合成了磁 - 等离子体激元纳米簇的关键步骤包括:拍摄高度单分散的初级金壳/氧化铁核心纳米颗粒和引导的一次粒子的自组装成纳米团簇。初级粒子和表面活性剂之间的摩尔比在确定的纳米团簇的尺寸分布中起重要作用。初级纳米颗粒的非均匀的粒度分布的磁 - 等离子体激元纳米簇组装期间导致形成大的聚集体。此外，纳米簇形成的微乳状液的方法依赖于两亲性表面活性剂:疏水性尾部基团持有初级纳米颗粒一起和亲水性头部基团稳定在水溶液中的纳米团簇。表面活性剂的浓度决定了纳米簇组装:高浓度会导致形成较小的簇或单独的初级颗粒和低浓度将导致粒子聚集。 <pCLASS ="jove_content"&gt;合成的纳米团簇具有从约 50纳米宽的粒径分布到约 300纳米，需要一个额外的分离步骤。如在协议中所述离心以逐渐增加的速度高于产生具有粒度分布的分离级分90±18纳米，130±26纳米，和180±39 nm的良好效果。更细的分离，以产生更窄的分布应该是可以通过使用尺寸排阻色谱法。还应该注意的是，纳米团簇具有在红色-近红外区域，提供了一个机会，以激发等离子共振用约 500至900纳米（ 图4）之间的任何来源的宽吸收。然而，这个属性也限制了纳米团簇适用性的多个目标同时成像。 的纳米团簇增加〜10-15纳米抗体结合后，流体力学半径。这种增加的直径相关WEL升与通过与Fc部分与纳米颗粒的表面附着的IgG抗体的约 12nm的尺寸。因此，在流体动力学直径的变化是与在该协议实现通过Fc部分的抗体的定向结合化学一致。纳米粒子的zeta电位转移，从-47.6 mV的抗体偶联到-7.0 mV的共轭前后。表面电荷的改变提供了抗体偶联到纳米团簇的其他证据。 本文所描述的协议的独特特征是合成各种尺寸从主块也具有磁电浆特性的磁 - 等离子体激元纳米颗粒。此方法提供了一种简单的方法来同时控制所得的纳米结构的表面等离子体和磁特性的强度。与此相反，以往的协议中使用电浆和磁性纳米材料的组装■一个材料曾担任另外一个沉积的模板在哪里;在这种方法中的一种材料占用量和所得到的纳米结构的另一表面上。文献报道的磁-等离子体激元纳米颗粒具有显著较低的密度和超顺磁性粒子的相比，由我们的协议14,15制成的纳米团簇的总量。在我们的方法和磁等离激元的部分在整个混合磁 - 等离子体激元纳米颗粒的量均匀地分布。

混合纳米粒子由不同的材料具有不同的理化性质，可在医学领域的应用，包括多模式分子影像学，治疗和交付的监控，新的筛选和诊断分析1-3打开新的机会。等离激元和磁特性的单个纳米颗粒的组合是特别感兴趣的，因为它提供了与等离子体激元共振和响应相关联的磁场很强的光散射和吸收截面。例如，磁-等离子体激元纳米颗粒，使用了经由外部电磁铁3-5施加时间信号调制，以增加在标记细胞的暗场成像的对比度。磁光声成像，在磁等离激元纳米粒子能在对比度和信后台大鼠很大的改进 - 最近，类似的原则在新的成像方法的发展施加IO 6,7。它还表明，该混合纳米粒子可用于同时捕获和检测全血中和体内 8,9循环肿瘤细胞。此外，磁-等离子体激元纳米颗粒是有希望的治疗诊断剂，其可用于分子特定的光学和磁共振成像结合癌细胞10的光热治疗。 有几种方法进行了探讨合成磁等离激元纳米粒子。例如，Yu 等人利用分解和Fe（CO）的氧化5金纳米粒子，形成哑铃状的双功能金， 四氧化三铁纳米粒子11。王等人已经用热分解方法12合成的涂覆金的氧化铁纳米颗粒。一些其他方法依赖于涂层聚合物或胺官能分子上磁心纳米颗粒随后AG的沉积旧壳到聚合物表面以产生杂种粒子7,13。此外，氧化铁纳米颗粒通过静电相互作用或发生化学反应14,15连接到金纳米棒。虽然这些方法中，得到磁 - 等离子体激元纳米结构，它们兼顾到磁电浆组合的某种程度的属性，例如光吸收在近红外（NIR）窗口或一个强大的磁力矩这两者都是在生物医学应用中是非常理想的。例如，哑铃金- 四氧化三铁纳米粒子在520nm处，限制其在体内的效用，由于高浊度的组织在这个光谱范围内的等离子体共振峰。此外，通过当前的协议所产生的磁-等离子体激元纳米颗粒被局限于一个11或少数（小于10），14,15超顺磁性基团（ 例如 ，氧化铁纳米颗粒）是显著小于可能是ACHieved在密密麻麻的纳米结构。例如，密密麻麻的直径60纳米的球形纳米颗粒可以包含上千6纳米超顺磁性纳米颗粒的数量级上。因此，存在对于改善混合纳米粒子的磁特性有很大空间。此外，一些先前所描述的协议是相对复杂的，需要以避免合成14,15中粒子的凝集仔细优化。 在这里，我们描述了一个协议，用于合成磁等离激元纳米粒子具有较强的磁矩和强大的近红外吸收，解决了目前先进的主要限制。合成有其油包水微乳液法16渊源。它是基于来自一个更小的一次粒子的纳米粒子所需的尺寸的组件。这种方法已被成功地用于生产由单一材料的纳米结构，例如金，铁氧化物和半导体的pri玛丽颗粒16。我们扩展它来合成磁电浆纳米颗粒的通过，第一，使得6纳米直径的金壳/氧化铁芯颗粒，然后，装配主杂种颗粒进入最后的球形纳米结构。组装一次颗粒的纳米团簇，不仅使增强组分的纳米颗粒，如实现更强的磁力矩，同时保持超顺磁性的特性，但还需要单独的纳米颗粒从而形成从构成的纳米粒子不存在的新的特性，如强之间的相互作用的优点光吸收的近红外窗口。这个协议产生混合纳米粒子与磁场和等离激元的功能的高密度。后一次粒子合成出，我们的方法本质上是一个简单的单罐反应。整体的等离子体共振的强度和磁矩是由一些初级粒子和，疗法的确定安伏，可以根据用途容易地优化。此外，我们还制定了抗体偶联过程的混合纳米粒子的各种生物医学应用程序需要特定的分子靶向。抗体通过与Fc部分离开Fab部分，它负责抗原结合可用于靶向附着。

<table><tbody><tr><td>PYREX 50 ml round bottom boiling flask with short neck &amp;amp; 24/40 [ST] joint</td><td>Corning</td><td>4320A-50</td><td>Thermal decomposition reaction</td></tr><tr><td>PYREX 41 x 300 mm 5-bulb Allihn condenser with 24/40 [ST] outer/inner joints</td><td>Corning</td><td>2480-300</td><td>Thermal decomposition reaction</td></tr><tr><td>Silicone oil</td><td>Fisher</td><td>S159-500</td><td>Oil bath</td></tr><tr><td>Hot plate stirrer</td><td>Corning</td><td>PC-351</td><td>Heat the reacton with stirring function</td></tr><tr><td>Thermometer</td><td>ThermoWorks</td><td>221-092</td><td>Measure temperature</td></tr><tr><td>Iron(III) acetylacetonate</td><td>Fisher</td><td>AC11913-0250</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Oleic acid 99%</td><td>Fisher</td><td>A195-500</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Gold(III) acetate</td><td>Fisher</td><td>AA3974206</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Hexane</td><td>Fisher</td><td>H292-1</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Phenyl ether 99%</td><td>Fisher</td><td>AC13060-0025</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>1,2-Hexadecanediol 90%</td><td>Sigma</td><td>213748-50G</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Oleylamine 70%</td><td>Sigma</td><td>O7805-100G</td><td>Material for primary hybrid nanoparticles synthesis</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Fisher</td><td>BP166-100</td><td>Cluster synthesis</td></tr><tr><td>Sodium citrate dihydrate</td><td>Sigma</td><td>W302600</td><td>Cluster synthesis</td></tr><tr><td>Monoclonal anti-EGF receptor antibody</td><td>Sigma</td><td>E2156</td><td>Cell labeling specificity test</td></tr><tr><td>Monoclonal anti-HER2 antibody</td><td>Sigma</td><td>AMAB90627</td><td>Cell labeling specificity test</td></tr><tr><td>Sodium periodate</td><td>Sigma</td><td>311448</td><td>Oxidate Fc region of antibodies</td></tr><tr><td>Dithiolaromatic PEG6-CONHNH&lt;sub&gt;2&lt;/sub&gt;</td><td>SensoPath Technologies</td><td>SPT-0014B</td><td>Heterofunctional linker for antibody conjugation to nanoclusters</td></tr><tr><td>Methoxy-PEG-thiol, 5 k</td><td>Creative PEGworks</td><td>PLS-604</td><td>Passivate the remaining gold surface after antibody conjugation</td></tr><tr><td>Amicon Ultra-4 centrifugal filter unit with Ultracel-10 membrane</td><td>Millipore</td><td>UFC801008</td><td>Protein purification</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td>Buffer</td></tr><tr><td>PBS, 1x solution</td><td>Fisher</td><td>BP2438-20</td><td>Buffer</td></tr><tr><td>UV-Vis spectroscopy</td><td>BioTek&amp;nbsp;</td><td>Synergy HT</td><td>Obtain spectrum</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>Separation</td></tr><tr><td>Transmission Electron Microscope</td><td>FEI</td><td>TECNAI G2 F20 X-TWIN</td><td>Obtain morphology of nanostructures</td></tr><tr><td>Upright microscope</td><td>Leica</td><td>DM6000</td><td>Obtain dark-field images</td></tr><tr><td>Sonicator</td><td>Branson</td><td>1510</td><td>Sonication</td></tr><tr><td>Carbon film 300 mesh grid</td><td>EMS</td><td>CF300-Cu</td><td>TEM imaging</td></tr><tr><td>96-well plate</td><td>Corning</td><td>09-761-145</td><td>UV-Vis reading plate</td></tr></tbody></table>

synthesis of immunotargeted magneto-plasmonic nanoclusters

磁性和电浆特性结合在一个单一的纳米颗粒提供了协同作用是有利的，在许多生物医学应用，包括在新的磁成像方式的对比度增强，同时捕获和检测（检验中心）的循环肿瘤细胞，以及多峰分子成像结合光热治疗的癌细胞。这些应用刺激了协议的发展为合成磁电浆纳米颗粒在近红外（NIR）区域的光吸收率和较强的磁矩的显著兴趣。这里，我们提出了一种新的协议，用于合成这种杂化的纳米颗粒是基于一种油包水微乳液的方法。本文所描述的协议的独特特征是合成各种尺寸从主块也具有磁电浆特性的磁 - 等离子体激元纳米颗粒。这种方法产生的纳米颗粒具有高登sity其中在整个所述纳米颗粒体积均匀分布的磁场和等离子官能团。该混合的纳米颗粒可以通过与Fc部分离开Fab部分，它负责抗原结合可用于靶向附着的抗体很容易地官能化。

甲方案为合成immunotargeted磁电浆纳米簇的示于图2，首先，磁性的Fe 3 O 4的氧化铁纳米颗粒是通过热分解法合成出。然后，一个薄约 1纳米金壳上沉积通过热分解氧化铁核心颗粒。主约 6纳米杂化的纳米颗粒作为种子，以通过利用一个油包水微乳液的方法制造磁-等离子体激元纳米团簇。纳米团簇被官能化的分子特异性靶向的单克隆抗体。 所合成的氧化铁核心纳米颗粒的尺寸为〜5纳米的直径。后金壳沉积在磁芯，初级氧化铁核/金壳纳米颗粒的尺寸增大到直径〜6纳米。从褐色胶体的颜色变化为氧化铁纳米颗粒到红紫色的金壳和沉积后，最后，对紫灰色的一次粒子的组装之后进入〜180nm的直径的球形纳米团簇（ 图3）。紫外-可见光谱表明主氧化铁芯/金壳纳米颗粒具有在530纳米的不存在于裸氧化铁颗粒（ 图4）独特的共振峰。当集群的形成，谱变化明显，并表现出强烈的宽广近红外吸收（ 图4）。 纳米团簇被缀合的单克隆抗体，以特异性靶向感兴趣的生物分子。缀合协议利用了杂聚乙二醇（PEG）接头，重视抗体的Fc区域的纳米团簇表面。连接器的一端有一个酰肼部分，其与氧化的糖基化的抗体部分进行交互。接头的另一端包括具有与纳米团簇的金表面具有强亲和力的二硫醇基团。以正在示威ê分子靶向我们选择的EGFR阳性的皮肤癌细胞株（A-431）和HER2阳性乳腺癌细胞株（SK-BR-3）。纳米团簇被官能化与抗EGFR或抗HER2抗体随后，分别与A-431或SK-BR-3肿瘤细胞混合。在图5中 ，一个明亮的金橙色的A-431和SK-BR-3肿瘤细胞表示分子特异性纳米团簇结合到相应受体的癌细胞。与此相反，不相关的聚乙二醇化的纳米团簇不与癌细胞相互作用。这些结果表明，功能化的纳米团簇分子的特异性。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/52090/52090fig1highres.jpg" width="500" /> 图1的实验装置用于合成主氧化铁核/金壳纳米颗粒的一个圆底烧瓶相连的冷凝器。反应<html>根据温度监测下进行的油浴中通过温度计。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52090/52090fig2highres.jpg" width="500" /> 图2示意性说明在合成immunotargeted磁等离激元纳米团簇的主要步骤。 <a href="https://www.jove.com/files/ftp_upload/52090/52090fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/52090/52090fig3highres.jpg" width="500" /> 图3的TEM图像和纳米颗粒的胶体悬浮液的颜色:（左）氧化铁纳米颗粒的核心; （中）镀金的氧化铁纳米颗粒; （右）混合磁等离激元纳米团簇。在透射电子显微镜图像比例尺为50纳米。<html> PS://www.jove.com/files/ftp_upload/52090/52090fig3highres.jpg"目标="_ blank将"&gt;请点击这里查看该图的放大版本。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/52090/52090fig4highres.jpg" width="500" /> 图4（A）的紫外-可见-近红外光谱的氧化铁核心纳米颗粒的光谱（蓝色），金涂覆的氧化铁纳米颗粒（绿色），和混合磁-等离子体激元纳米团簇（红色）（B）紫外-可见-近红外光谱用各种尺寸的混合磁 - 等离子体激元纳米簇:90纳米（蓝），130纳米（绿）和180纳米（红色）。所有光谱归一，在最大吸收，以显示光谱曲线差异。 <a href="https://www.jove.com/files/ftp_upload/52090/52090fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a><html> ANK"&gt;请点击这里查看该图的放大版本。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/52090/52090fig5highres.jpg" width="500" /> 图5的抗体偶联磁等离激元纳米团簇分子特异性:（左）表皮生长因子受体表达与培养EGFR靶向纳米团簇的A-431皮肤癌细胞; （中）表达HER2的温育与HER-2靶向纳米团簇SK-BR-3乳腺癌细胞; （右）A-431细胞培养与不相关的聚乙二醇化纳米团簇。细胞的黄橙色的颜色指示了由官能化的纳米团簇成功的标记;灰蓝色的颜色对应于从细胞中内源的散射。采用立式显微镜20X暗场物镜和氙灯激发被收购的图像。比例尺为10微米。_blank"&gt;请点击这里查看该图的放大版本。 <a href="https://www.jove.com/files/ftp_upload/52090/Video_Figure_1.m4v" target="_blank">影片1。</a>该视频比较标记通过任一初级纳米颗粒或纳米簇，以外部磁场的A-431肿瘤细胞的应答。其中结合有用于EGFR（+）A431细胞的特异性靶向抗EGFR抗体既粒子类型。首先，将微量离心管中填充标记的细胞悬浮液。然后，磁铁放在旁边的管细胞的运动进行成像，在约 10毫米远离磁铁。在左侧的动画显示标记的细胞主要纳米颗粒（直径为6纳米）和右边的电影 - 标记的细胞的磁电浆纳米团簇（直径为100纳米）。用明视场模式的倒置显微镜20倍的目标被收购的电影。比例尺为100微米。 </p&gt;

Watch this Scientific Journal Video about Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters at JoVE.com

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

在这里，我们描述了一个协议，用于合成磁等离激元纳米粒子具有较强的磁矩和强大的近红外（NIR）吸收。该协议还包括抗体缀合，以通过与Fc部分关于各种生物医学应用的需要分子特异性靶向的纳米颗粒。

Immunotargeted的合成磁等离子团簇

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

synthesis-of-immunotargeted-magneto-plasmonic-nanoclusters

Nanyang Technological University

Research

JoVE Journal

Chemistry

15.2K 次观看.  University of Texas at Austin. 在这里，我们描述了一个协议，用于合成磁等离激元纳米粒子具有较强的磁矩和强大的近红外（NIR）吸收。该协议还包括抗体缀合，以通过与Fc部分关于各种生物医学应用的需要分子特异性靶向的纳米颗粒。 

视频: Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

为增强微型和纳米操纵利用电浆和光子晶体纳米结构

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

科研

生物工程

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

合成，表征和混合金/硫化镉和Au / ZnS核/壳纳米颗粒功能化

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

化学

Multifunctional Hybrid Fe2O3-Au Nanoparticles for Efficient Plasmonic Heating

One of the most widely used methods for manufacturing colloidal gold nanospherical particles involves the reduction of chloroauric acid (HAuCl4) to neutral gold Au(0) by reducing agents, such as sodium citrate or sodium borohydride. The extension of this method to decorate iron oxide or similar nanoparticles with gold nanoparticles to create multifunctional hybrid Fe2O3-Au nanoparticles is straightforward. This approach yields fairly good control over Au nanoparticle dimensions and loading onto Fe2O3. Additionally, the Au metal size, shape, and loading can easily be tuned by changing experimental parameters (e.g., reactant concentrations, reducing agents, surfactants, etc.). An advantage of this procedure is that the reaction can be done in air or water, and, in principle, is amenable to scaling up. The use of such optically tunable Fe2O3-Au nanoparticles for hyperthermia studies is an attractive option as it capitalizes on plasmonic heating of gold nanoparticles tuned to absorb light strongly in the VIS-NIR region. In addition to its plasmonic effects, nanoscale Au provides a unique surface for interesting chemistries and catalysis. The Fe2O3 material provides additional functionality due to its magnetic property. For example, an external magnetic field could be used to collect and recycle the hybrid Fe2O3-Au nanoparticles after a catalytic experiment, or alternatively, the magnetic Fe2O3 can be used for hyperthermia studies through magnetic heat induction. The photothermal experiment described in this report measures bulk temperature change and nanoparticle solution mass loss as functions of time using infrared thermocouples and a balance, respectively. The ease of sample preparation and the use of readily available equipment are distinct advantages of this technique. A caveat is that these photothermal measurements assess the bulk solution temperature and not the surface of the nanoparticle where the heat is transduced and the temperature is likely to be higher.

Multifunctional Hybrid Fe2O3-Au Nanoparticles for Efficient Plasmonic Heating

We describe the synthesis and properties of multifunctional Fe2O3-Au nanoparticles produced by a wet chemical approach and investigate their photothermal properties using laser irradiation. The composite Fe2O3-Au nanoparticles retain the properties of both materials, creating a multifunctional structure with excellent magnetic and plasmonic properties.

多功能混合铁<sub&gt; 2</sub&gt; 0<sub&gt; 3</sub&gt; -Au纳米粒子高效的电浆暖气

One of the most widely used methods for manufacturing colloidal gold nanospherical particles involves the reduction of chloroauric acid (HAuCl4) to ...

工程学

Colloidal Synthesis of Nanopatch Antennas for Applications in Plasmonics and Nanophotonics

We present a method for colloidal synthesis of silver nanocubes and the use of these in combination with a smooth gold film, to fabricate plasmonic nanoscale patch antennas. This includes a detailed procedure for the fabrication of thin films with a well-controlled thickness over macroscopic areas using layer-by-layer deposition of polyelectrolyte polymers, namely poly(allylamine) hydrochloride (PAH) and polystyrene sulfonate (PSS). These polyelectrolyte spacer layers serve as a dielectric gap in between silver nanocubes and a gold film. By controlling the size of the nanocubes or the gap thickness, the plasmon resonance can be tuned from about 500 nm to 700 nm. Next, we demonstrate how to incorporate organic sulfo-cyanine5 carboxylic acid (Cy5) dye molecules into the dielectric polymer gap region of the nanopatch antennas. Finally, we show greatly enhanced fluorescence of the Cy5 dyes by spectrally matching the plasmon resonance with the excitation energy and the Cy5 absorption peak. The method presented here enables the fabrication of plasmonic nanopatch antennas with well-controlled dimensions utilizing colloidal synthesis and a layer-by-layer dip-coating process with the potential for low cost and large-scale production. These nanopatch antennas hold great promise for practical applications, for example in sensing, ultrafast optoelectronic devices and for high-efficiency photodetectors.

A protocol for the colloidal synthesis of silver nanocubes and fabrication of plasmonic nanoscale patch antennas with sub-10 nm gaps is presented.

在等离子体激元和纳米光子学应用Nanopatch天线的合成胶体

We present a method for colloidal synthesis of silver nanocubes and the use of these in combination with a smooth gold film, to fabricate plasmonic ...

Fabrication of Periodic Gold Nanocup Arrays Using Colloidal Lithography

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing in combination with new nanofabrication techniques. A plasmon is a quantum of charge density oscillation that lends nanoscale metals such as gold and silver unique optical properties. In particular, gold and silver nanoparticles exhibit localized surface plasmon resonances-collective charge density oscillations on the surface of the nanoparticle-in the visible spectrum. Here, we focus on the fabrication of periodic arrays of anisotropic plasmonic nanostructures. These half-shell (or nanocup) structures can exhibit additional unique light-bending and polarization-dependent optical properties that simple isotropic nanostructures cannot. Researchers are interested in the fabrication of periodic arrays of nanocups for a wide variety of applications such as low-cost optical devices, surface-enhanced Raman scattering, and tamper indication. We present a scalable technique based on colloidal lithography in which it is possible to easily fabricate large periodic arrays of nanocups using spin-coating and self-assembled commercially available polymeric nanospheres. Electron microscopy and optical spectroscopy from the visible to near-infrared (near-IR) was performed to confirm successful nanocup fabrication. We conclude with a demonstration of the transfer of nanocups to a flexible, conformal adhesive film.

We demonstrate the fabrication of periodic gold nanocup arrays using colloidal lithographic techniques and discuss the importance of nanoplasmonic films.

用胶体光刻制备周期性金 Nanocup 阵列

Within recent years, the field of plasmonics has exploded as researchers have demonstrated exciting applications related to chemical and optical sensing ...

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

利用磁性纳米粒子丰富和扩大罕见抗原特异性 t 细胞

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

细胞标记和超顺磁性氧化铁纳米粒子靶向

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical lattice consists of an array of nanostructures, which exhibit a variety of trapping and transport behaviors. We report the experimental procedures to trap micro-particles in a simple square nanoplasmonic optical lattice. We also describe the optical setup and the nanofabrication of a nanoplasmonic array. The optical potential is created by illuminating an array of gold nanodiscs with a Gaussian beam of 980 nm wavelength, and exciting plasmon resonance. The motion of particles is monitored by fluorescence imaging. A scheme to suppress photothermal convection is also described to increase usable optical power for optimal trapping. Suppression of convection is achieved by cooling the sample to a low temperature, and utilizing the near-zero thermal expansion coefficient of a water medium. Both single particle transport and multiple particle trapping are reported here.

We describe a procedure to optically trap micro-particles in nanoplasmonic optical lattice.

Nanoplasmonic 光学晶格中微粒子的俘获

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

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 ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

磁性、声学和光学三响应性微泡，用于磁性高温和波托托热组合癌症治疗

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...