JMD公司和MKB的资助通过IGERT奈米科学和技术方案，在东北大学（资金来自美国国立癌症研究所和美国国家科学基金会资助DGE - 0504331）。这项工作是由美国国立卫生研究院国家普通医学科学格兰特R01 GM084366研究院资助。我们也感谢Saumya Das和安东尼BIDMC罗森茨维格在波士顿为他在动物协议的发展作出贡献的心肌细胞和凯文现金。

1。准备的optode 等分的组件的optode之前，都需要这样，他们可以很容易地测量和存储。 <ol><li>一个50毫克的钠离子载体X（NaIX）小瓶和50毫克钠四 - [3,5 - 双（三氟甲基）苯基]硼酸（NaTFPB）将各自提出了在tetrahyrdofuran（THF）和分装到1.5毫升聚苯乙烯离心管。在化学通风柜，溶于四氢呋喃在1毫升50毫克的NaIX航运瓶和组合，以确保完全溶解固体。转移到10个不同的离心管，100μL该解决方案。重复NaTFPB。允许的THF在化学通风柜一夜之间蒸发，标签的离心管，并将其放置在一个存储4度冰箱。这将创建5毫克等分干NaIX和NaTFPB。 </li><li> Chromoionophore III（CHIII）溶解于1毫升四氢呋喃，并转移到了3毫升的玻璃小瓶。另1毫升四氢呋喃CHIII航运小瓶解散任何残留CHIII，并加入3毫升玻璃瓶。将有一个目前共有2毫升。将1毫升的四氢呋喃溶液中的CHIII两个单独的1.5毫升，用聚四氟乙烯涂盖的玻璃小瓶。商店在4度的冰箱在终浓度为5毫克/毫升的CHIII的解决方案。 </li></ol>这些分装和解决方案将被用来制造optode材料。 <ol start="3"><li>移液器66μL之二（2 - 乙基己基）癸二（DOS）到1.5 ml的聚四氟乙烯涂层螺丝帽的玻璃小瓶 - 这是optode小瓶。 DOS是一种粘稠的解决方案，因此必须小心，以确保适量的解决方案，是转移到小瓶。该卷对应〜60毫克的DOS。 </li><li>称取30毫克超高分子量聚（乙烯基）聚氯乙烯（PVC）和转移optode小瓶。 </li><li>现在添加optode小瓶分装功能部件，以创建所需的的optode。 CHIII，NaIX，并NaTFPB的相对浓度，确定传感器的钠的反应。这些浓度可以调整到理想的响应细胞内的浓度，与10毫米，或胞外浓度KD与150毫米KD，有一个传感器。此外，还有可能会被一批批从供应商和校准传感器的化学品的变异性是必要的，以确定是否正确的反应是为每一批新的化学成分发生。在这里，我们描述的方法来创建一个传感器通常用于测量细胞内钠的浓度。 </li><li>在化学通风柜，转移100μL5毫克/毫升CHIII的四氢呋喃溶液optode小瓶。 </li><li>溶解于300μL四氢呋喃NaIX 5毫克和300μL转移到optode小瓶，这关联到5毫克NaIX。 </li><li>溶解于500μL四氢呋喃的5毫克的NaTFPB等分和20μL的溶液转移到optode小瓶，此关联NaTFPB 0.1毫克。 </li><li>加入80μL四氢呋喃optode小瓶。 </li><li> optode小瓶溶液中含有30毫克的PVC，DOS的60毫克，5毫克，0.2毫克NaTFPB，CHIII 0.5毫克，500μL四氢呋喃NaIX。直到所有的PVC瓶轻轻涡旋这已经解散。 optode应该是红色。四氢呋喃总额添加到小瓶，不管使用的组件的比例应为500μL。 </li><li>允许NaIX和NaTFPB等分余下的四氢呋喃一夜之间蒸发和重新标签管的化学品的正确。 </li></ol> 2。创建的纳米传感器<ol><li>加入100μL10 mg / ml的1,2 - Distearoyl - SN -甘油3 Phosphoethanolamine - N - [甲氧基（聚乙二醇）-550]氯仿（聚乙二醇脂质）到4 DRAM的玻璃小瓶。允许氯仿蒸发在化学通风柜。这个过程可以加快干燥氮气或内部空气的解决方案。 </li><li>添加4毫升的小瓶PEG脂质所需的水溶液。超声提示下面的小瓶放置在实验室插孔和提高小瓶，直到头大约7毫米深的解决方案，低超声功率超声为30秒。在这里，我们使用一个布兰森数字sonifier一个半英寸的尖端直径一步喇叭设置15％的幅度。水溶液可以是任何解决方案。例如，要确定我们trizma基地使用10毫米的HEPES pH值7.2的传感器的响应。 </li><li>虽然解决方案正在超声，50μL与50μL的二氯甲烷在0.6毫升的离心管optode材料混合。用移液管组合，以确保混合均匀。 </li><li>调整sonifier控制超声3分钟。启动超声，而超声添加到溶液中浸泡枪头和分配在一个快速，甚至注射液100μLoptode混合溶液的小瓶。该解决方案应该变成蓝色，depending对水的使用解决方案。 </li><li>允许的超声持续约30秒，然后降低超声提示，使溶液中的约2毫米深的插孔。这应该开始通气的解决方案和解决方案将变得不透明。这一步，​​有助于消除从溶液中剩余的四氢呋喃和二氯甲烷。 </li><li>超声是完成后，拉起到5毫升注射器的纳米传感器的解决方案。附加的0.2微米注射器的过滤器和分配到一个螺丝顶盖的玻璃小瓶纳米传感器解决方案。该解决方案应该是明确的，蓝色的，如果使用水溶液中包含小于10毫米的钠离子和pH值小于约9。否则，它应该没有颜色，是粉红色的。 </li></ol> 3。确定nanonsensor回应使用此方法来确定的，旨在衡量细胞内钠的纳米传感器的响应。细胞外钠离子浓度的纳米传感器用于不同浓度的建议。 <ol><li> 10毫米的HEPES（0 NA）和1M氯化钠（氯化钠）在10毫米的HEPES（1米娜）和pH值这些解决方案的每个7.2使用trizma基地解决方案。混合在50毫升锥形离心管娜0娜和1个M的库存解决方案来创建解决方案：0，1，5，10，20，50，100，200，500，1000毫米的NaCl浓度。 </li><li>使用光学底部的96孔板中，以确定响应。添加100μL，每个浓度的钠一列3口井。这将产生一个3 × 10口井的阵列。 </li><li>每孔加100μL新鲜配制的纳米传感器，并加载到微孔板荧光。我们使用一个分子器件Spectramax供应M3的。 </li><li>每孔读以下的波长： </li></ol><table border="1"><tbody><tr><td> 激励（NM） </td><td> 排放量（纳米） </td><td> 切断（NM） </td></tr><tr><td> 488 </td><td> 570 </td><td> 530 </td></tr><tr><td> 488 </td><td> 670 </td><td> 610 </td></tr><tr><td> 639 </td><td> 680 </td><td> 665 </td></tr></tbody></table>在每个波长的纳米传感器强度是依赖于钠离子浓度。使用的波长取决于应用程序。细胞内缓慢的动态测量，我们使用488 nm/570纳米（激发/发射）和488 nm/670 nm，这让我们两个波长的比例和降低噪音。 <ol start="5"><li>传统上，optode数据被转换为一个值，这是：α=（我- 我分钟）/（I MAX - 我分），我是在一个给定的的钠离子浓度的强度 ，我分是在响应测量的最低强度我最大的是中强度最高的响应测量。转换的强度比在570 nm处，除以在670 nm处的强度或强度在680 nm的α。 </li><li>使用绘图软件，无论是Excel或原产地通常在我们的实验室中使用，情节与钠离子浓度的日志α，钠离子浓度为零的记录设置为-1。一个S形曲线拟合响应。从曲线，计算在α= 0.5的钠离子浓度，代表浓度传感器的最佳响应。 </li><li>调整CHIII，NaIX，并NaTFPB的比例，在optode创建最佳反应，与周围的利益钠离子浓度的纳米传感器。 </li></ol> 4。细胞内成像<ol><li>细胞内的测量，纳米传感器在5毫克/毫升的葡萄糖水，进入细胞内注射。 </li><li>细胞生长或转移到玻璃底部盘或玻璃盖玻片底部的成像室。 </li><li>明确的媒体或台氏液培养的细胞。装入到一个epifluorescence显微镜成像室或菜。我们使用的Zeiss LSM的7共聚焦显微镜。 </li><li>毛细玻璃吸管拉马拉。我们使用了1毫米外径，0.78 mm内径玻璃萨特燃烧棕P - 97，到最后尖端直径约300-500纳米。 </li><li>回填与纳米传感器，使用注射器和汉密尔顿针头的解决方案，或者一个微尖器和吸管吸管。 </li><li>吸液管安装到一个是连接到一个显微操纵器和连接到压力控制喷射系统的持有人。在这里，我们使用一个伯利EXFO的PCS 6000显微和医疗系统公司的喷油器。 </li><li>降低的细胞上的吸管，进入细胞质中，有时是必要的“龙头”操盘人捅破了膜。注入的纳米传感器的解决方案，并迅速撤回移液器。 </li></ol> 5， 在活体成像 所有程序已经由东北大学的动物护理和使用委员会的审查和批准 。 <ol><li>纳米传感器在体内 ，外成像磷酸盐缓冲液，并在135毫米的钠离子浓度调整到最佳响应钠。 </li><li>对于注射的设置和在小鼠体内的荧光纳米传感器的成像，我们使用IVIS Lumina的第二和其关联麻醉单位。消毒的感应和成像商会。放置在感应室CD1的裸鼠（约20克），并使它们为异氟醚。％异氟醚和氧气流量管理上的物种和小鼠的体重会有所不同而有所不同。等待的老鼠变得完全麻醉。 </li><li>小鼠被麻醉后，从诱导室和地方删除成像室的老鼠。保持在麻醉状态下的小鼠。对于每个鼠标，消毒所需的注射区。 </li><li> 10μL的纳米传感器，确保删除所有气泡填充31G无菌胰岛素注射器。 </li><li>使用镊子，把握沿所需的注射部位在鼠标背面的一小块皮肤倍。在抓皮肤，插入皮肤注射器朝上的斜面和皮肤针平行。 </li><li>注射传感器之前，拉注射器推杆回，以确保不进针插入血管。然后，轻轻移动针左，右创建一个皮肤内的口袋里。这将最大限度地减少回压，从而导致泄漏注射部位的传感器。注入的传感器，轻轻取下注射器。 </li><li>轻轻施加压力，注射部位，擦干注射部位泄露任何可能的解决方案。这将最大限度地减少不注射到皮肤的传感器的荧光。 </li><li>重复步骤，直到所需的注射区的数量达到5.4至5.7。六注射液均匀地分布在背面的左侧和右侧间隔建议。对于每个单独的鼠标，改变针针插入皮肤变得很困难。 针不应该小鼠之间共享，这将最大限度地减少疾病的传播或在小鼠中的交叉污染。 </li><li>成像的钠荧光传感器，我们获得了使用过滤器设置最紧密匹配的640 nm/680纳米的纳米传感器（激发/发射）频谱，也可以减少皮肤的自体荧光的小鼠明和荧光图像。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/2896/2896fig1.jpg" alt="图1"/> 图1五种不同的纳米传感器套钠的响应曲线。每套代表从不同optode的配方CHIII，NaTFPB，PVC和DOS相同数额，但NaIX数额不等的纳米传感器。数据被转换为使用680分之639纳米强度的α值。数据是平均3，错误为清晰起见，省略了酒吧，与使用原产一个装有S形曲线。在此响应曲线，使用钠的最高浓度是500毫米。 KD钠钠纳米传感器不等，从10毫米（橙色钻石）到150毫米（黑色方块）。 <img src="/files/ftp_upload/2896/2896fig2.jpg" alt="图2"/> 图2。注入新生的心肌细胞。细胞培养上的玻璃罩，安装在显微镜上，并与钠的纳米传感器注射。显示荧光（639 nm激发），明，和覆盖。请注意，一些传感器集群出现，但细胞形态正常，传感器已经扩散整个细胞质中，并没有核装载。 <img src="/files/ftp_upload/2896/2896fig3.jpg" alt="图3"/> 图3。皮下注射小鼠钠纳米传感器。 9钠纳米传感器的不同注射到裸鼠的皮下空间。明场和荧光图像（六百八十零分之六百四十）重叠。

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应采取不再形成的纳米传感器已经取得了超过10分钟，一旦optode解决方案和PEG脂质干。 Optode可以不仅在需要时迅速，但在4摄氏度存储时，它们可以几个月的稳定。我们已经表明，nansensors一旦形成，在溶液中稳定，至少一个星期;但是，必须小心，以防止阻止光的纳米传感器，超过这个时间的漂白6虽然钠的纳米传感器在这里进行了论证，已optodes建立生物物种最为相关的离子，如钾和氯化物。10,12，我们还扩大这项技术的小分子如葡萄糖13 。 除了生成的纳米传感器，以监测不同分析物，这些纳米传感器适合其他的变化，这将使他们在大多数生物实验中的有用。例如，表面涂层，在这种情况下，聚乙二醇（PEG），可以很容易地改变使用任何双亲分子，易溶于水。这使functionalizing这些纳米粒子为不同的应用程序或目标的可能性。纳米粒子的大小也可以调整，通过改变表面涂层，超声强度或使用的溶剂。 钙荧光指标已在决定细胞内钙信号的宝贵;然而，没有可用的钠敏感染料具有相同的理想特性。 Optode纳米粒子成像离子细胞在多年的发展，提供了一个替代方法。14，我们已经呼吁建立此以前的研究创建特定的纳米传感器，细胞内的钠成像技术，希望能提供一种手段，了解如何钠信号影响细胞功能和在信号改变，从而导致某些疾病。 在体内的荧光纳米传感器的使用提供一个实时的监测分析物的替代微创超过其他方法 ，如抽血，3钠和葡萄糖纳米传感器基于上述技术已被证明是跟踪钠和葡萄糖的变化，分 ​​别在体内 。3,13然而，目前存在的局限性，这种方法作为一个监测工具。例如，传感器必须包含与对近红外红充分转移，以尽量减少从皮肤背景的自发荧光。15秒频谱荧光团，电流注入技术不产生均匀注射等可能造成的纳米传感器错误注射深度的变化。尽管有这些限制，这些荧光纳米传感器的发展和他们的成功示范，使这些传感器非常宝贵的研究工具和用于监测病人的健康的方法。

<table border="1"><tbody><tr><th>产品名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> IVIS Lumina的II仪器包</td><td>卡尺生命科学</td><td> 126273 </td><td></td></tr><tr><td> CD - 1裸鼠</td><td>查尔斯河</td><td>应变代号：086 </td><td>免疫缺陷</td></tr><tr><td>胰岛素注射器</td><td>屋宇署</td><td> 328438 </td><td> 3 / 10 毫升 毫升 ，8MM，31G </td></tr><tr><td>高相对分子重量聚氯乙烯</td><td>西格玛</td><td></td><td></td></tr><tr><td> DOS </td><td>西格玛</td><td></td><td></td></tr><tr><td> CHIII </td><td>西格玛</td><td></td><td></td></tr><tr><td> NaTFPB </td><td>西格玛</td><td></td><td></td></tr><tr><td> NaIX </td><td>西格玛</td><td></td><td></td></tr><tr><td>薄壁毛细管玻璃</td><td>萨特</td><td></td><td></td></tr><tr><td>聚乙二醇脂质</td><td>阿凡提极地血脂</td><td></td><td></td></tr></tbody></table> 表1提供了一个在此过程中使用的化学品和设备的清单。未列出的所有常见的化学品均购自Sigma。

fluorescent nanoparticles for the measurement of ion concentration in biological systems

严格监管整个人体的离子平衡是必要的预防脱水等衰弱状态。相比之下，在细胞水平上的快速离子通量发起兴奋细胞动作电位的要求。2钠调节起着重要的作用既这些情况，但是，没有方法，目前存在的连续监测在体内 3和细胞内的钠探头 4没有提供类似的详细结果为钙探针的钠含量。在努力填补这些空洞，荧光纳米传感器已被开发，它可以监控在体外和体内的钠浓度。5,6这些传感器是基于离子选择性optode技术，包括塑在这种特定的识别钠的聚合物颗粒嵌入式元素，pH敏感的荧光，和添加剂。7-9机械，钠识别元素提取到的传感器钠10提取原因的pH敏感的荧光基团释放出氢离子，以保持电中性，这会导致传感器在荧光的变化。钠传感器比钾和钠是可逆的选择性，即使在细胞内浓度高6，他们是直径约120纳米和聚乙二醇涂层传授的生物相容性。 11此外，他们还实时跟踪体内钠浓度的变化时，注入利用显微注射技术，传感器可以传送到他们已被证明是监测时间和空间上跳动的心肌细胞钠动力学细胞的细胞质中。到小鼠皮下3这里，我们详细解释和展示的方法，用于制造荧光钠纳米传感器，并简要展示生物应用，我们的实验室使用的纳米传感器：微量注射到细胞中的传感器和传感器皮下注射入小鼠。

Watch this Scientific Journal Video about Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems at JoVE.com

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

我们的实验室中产生的荧光纳米颗粒用于成像的离子浓度和离子流，在生物系统，如在信令和组织间液的细胞在生理动态平衡。

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

fluorescent-nanoparticles-for-measurement-ion-concentration

Research

JoVE Journal

Bioengineering

15.2K 次观看.  Northeastern University. 我们的实验室中产生的荧光纳米颗粒用于成像的离子浓度和离子流，在生物系统，如在信令和组织间液的细胞在生理动态平衡。

视频: Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

科研

生物工程

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

在小角度中子衍射仪KWS-2研究软物质和生物系统在广泛的长度尺度从纳米，微米尺寸

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

The protocol for a novel ion concentration polarization (ICP) platform that can stop the propagation of the ICP zone, regardless of the operating conditions is described. This unique ability of the platform lies in the use of merging ion depletion and enrichment, which are two polarities of the ICP phenomenon.

并列的合并离子交换膜之间的离子浓差极化，以块极化区的传播

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP ...

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

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

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 ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

闪速纳米沉淀法包聚物纳米粒子中疏水亲水化合物的作用

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli such as physical movement, activation of tactile sensors in the skin, and viewing images. Neural activation is inferred from an increase in the cerebral blood flow velocity (CBFV) supplying the region of the brain involved in processing sensory input. For example, viewing bright light causes increased neural activity in the occipital lobe of the cerebral cortex, leading to increased blood flow in the posterior cerebral artery, which supplies the occipital lobe. In fTCD, changes in CBFV are used to estimate changes in cerebral blood flow (CBF).
With its high temporal resolution measurement of blood flow velocities in the major cerebral arteries, fTCD complements other established functional imaging techniques. The goal of this Methods paper is to give step-by-step instructions for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying the middle cerebral artery (MCA) and optimizing the signal will be described. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is a specific example of a functional imaging experiment using fTCD, will be demonstrated.

Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment.&#160;

功能颅内多普勒超声监测脑血流

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 &#181;g of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

The study describes a protocol for creating large (&#181;g-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

使用无细胞系统对用于蛋白质原型的最小线性模板进行扩增的快速酶促方法

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...