这项工作是由国家研究资源中心和国家中心推进转化科学，国家卫生研究院，通过格兰特UL1TR000124（至FW）的支持;费利克斯和米尔德里德业明德教授和巴恩斯家庭基金（以DTWW），国家研究所美国国立卫生研究院的牙科及颅面研究下奖号码T90DE022734（以MT）。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

1.基于磁珠的外来体提取<ol><li>吸管5微升链霉亲和涂层的磁性体微粒的充分混合的溶液加入495微升磷酸盐缓冲盐水（PBS）缓冲液中离心管，以重悬珠子。用磁力架洗，重悬珠用500μlPBS中的3倍。机架是磁体上的壳体单元，可容纳的样品的离心管的侧阵列。 <ol><li>对于每一个洗，先让管子坐在架1分钟，然后用枪头小心地取出上清缓存，而不会干扰珠。 </li><li>放置在常规机架管子而不磁铁在侧。添加500微升PBS入管，并使用移液管以混合溶液和珠在一起。然后把管背面磁性机架上再次从溶液中分离磁珠。 </li><li>通过磁化和再悬浮在PBS中执行此清除缓冲区总共三次。此进行磁性粒子的初始洗涤。 </li></ol></li><li>重悬珠子到490微升的PBS缓冲液中，与管放置在磁性支架的非磁化部。吸管5微升生物素化的小鼠抗人CD63抗体以1.0mg / ml储备浓度成珠的混合物。使用吸移管以混合珠和抗体在溶液中。 </li><li>将离心管与珠和生物素标记抗体的混合物样品上肩。设置的旋转器参数为往复旋转样品旋转器，在90°倾斜5秒，并在5℃振动1秒。旋转样品珠混合物在管的这些参数在室温30分钟。 </li><li>结合后，除去未结合的抗体。 <ol><li>以下旋转在室温30分钟后，将管背磁架中5分钟。 </li><li>通过使用微量吸管除去液相执行珠粒洗涤三次并清洗用500μ;升PBS的。三重洗涤后，悬浮于机架的非磁化部分在490微升酪蛋白的PBS和地点的珠子。 </li></ol></li><li>外来体提取使用抗体包被的珠子。 <ol><li>标记每个管有针对性的样品ID。吸取血清或唾液的10微升样品放入离心管中。使用吸管吹打几次以混合样品和磁性珠粒。 </li><li>放置与旋转器样品和抗人CD63抗体的珠子的管子和旋转2小时，在室温。如在步骤1.2中所述使用相同的旋转器的参数。 </li><li>以下的2个小时的样品旋转的，由磁化分离珠从溶液中去除液体相用微量吸管，并在500微升的Tris-HCl缓冲液重悬珠子进行三重洗涤。所得珠粒现在结合到外来体，并准备对电场释放和测量。 </li></ol></li></ol> 2.电场感应发布了次外体含量的测定<ol><li>电极的初始与预涂底漆GADPH <ol><li>适用于塑料以及一个电极阵列，以防止交叉污染的个别电极。对于这个实验，使用16传感器电极阵列与每个单元的电极由一个工作，计数器，和参考制成的裸金电极的阵列中。 </li><li>吹打库存试剂与超纯蒸馏水的管制备股票混合物100nM的DNA探针，0.3M KCl和10mM的吡咯。涡旋调匀。 注:在本研究中，所选择的DNA探针对应于GAPDH基因的参考，这是公知的外来体中存在。所使用的探针序列是:5&#39;-生物素 - AGGTCCACCACTGACACGTTG-3&#39;。上所有的电极使用此混合物。 </li><li>吸管上各金电极的表面60微升单体-DNA探针混合物。检查电极，以确保有工作角的足够的覆盖范围ounter，和参考电极由液体混合物。 </li><li> Electropolymerize单体探针混合物通过施加环状方波（CSW）电场分布到电极表面，以创建在电极表面上的导电聚合物层。该电场由申请350 mV的9秒，并立即切换到950毫伏1秒。应用此环状方波剖面向电极对10个循环，共100秒施加的电场的。 </li><li>冲洗传感器表面3次，用蒸馏水和干氮气以从电极表面除去液体。确保液体从电极适当除去。 </li></ol></li><li>切体卸货<ol><li>负载5微升1μM的一个探测器探测到495微升珠外来体复合物的混合物，并使用移液管混合。 注:检测探针是偶联于荧光素分子在3&#39;末端的DNA引物序列。该探测器概率用于此留学e序列对应于外来体中找到的GAPDH的mRNA。荧光素缀合的检测探针的序列是:5&#39;-GCAGTGGGGACACGGAAGGCC荧光素-3&#39;。 </li><li>吸管上与下一个磁体阵列金电极表面60微升探针​​和珠子的外来体复合物的混合物。这种磁体阵列由排列以对应于所述传感器的工作电极162.54毫米直径的钕磁铁。 图2A示出的放置在磁铁和珠外来体溶液。 </li><li>一旦样品在电极表面上加载，应用20个周期的CSW电场与9秒，在-300毫伏和1秒，200毫伏（200秒计）。被释放的外来体的货物将杂交到引物的电极的表面上。如果外来体的表面标记是调查对象，跳过实验的这一部分。 图2B示出了这个过程。 </li> &lt;LI&gt;洗掉由三重漂洗电极表面用蒸馏水在电极表面上的未结合的分析物。干燥氮气的电极。 </li></ol></li><li>记者抗体和读出<ol><li>加入60微升150单位/ ml的抗荧光素抗体缀合到辣根过氧化物酶（在1 HRP:1,000稀释度）稀释于酪蛋白/ PBS中。 </li><li>使用电场驱动的缀合复合物的抗荧光素HRP到探针三明治。申请-200毫伏为1秒和500毫伏，1秒，5个循环到电极表面上。 图2A示出了用于两个蛋白质和核酸的系统的捕获和检测探针复合物。 </li><li>使用蒸馏水和干氮气三重洗涤传感器表面。 </li><li>继洗掉未结合的多余的抗荧光素抗体的，加入60微升的3,3&#39;，5,5&#39;-四甲基联苯胺（TMB）底物。装载在该基板上，以使用多通道移液管的每个传感器的表面上。 </li><li>通过测量电极的电流在-200毫伏为60秒，使用能够16路同时测量的电化学电位执行当前的安培读数。 图2C是在读出期间电流分布的一个例子。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/52439/52439fig2highres.jpg" /> 图2.组件EFIRM方法。（A）中提取使用抗人类CD63包被的磁性微粒生物流体外来体，然后使用施加到颗粒外来体复合物的循环方波卸货物外来体的方法。电极的生物传感器的（B）的计划用于从释放外来体检测的RNA / DNA /蛋白质的目标。 （C）的距离EFIRM方法论，安培读出的代表性的例子，其中较大的电流幅度将对应吨Ø更高水平的生物分子。这个数字是从威等14 <a href="https://www.jove.com/files/ftp_upload/52439/52439fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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王守业是RNAmeTRIX公司是一家分子诊断公司的创始人之一。 PeriRx LLC分许可有关的RNAmeTRIX分子诊断知识产权。王守业是一个顾问PeriRx。

其结果表明，抗 - 人CD63包被的磁性纳米颗粒能够特异性捕获具有的尺寸范围为70-100纳米的小颗粒。这个捕获粒子是与外来体的先前观察到的轮廓相一致。此外，低电压的CSW以下，颗粒的捕获的使用被示为从胎圈表面除去它们，并导致DNA降解型材类似于传统的裂解缓冲液基础的方法对货物的释放。此数据表明，外来体货物释放的工作流可以通过一个环状的方波的外来体的脂质膜的破坏的应用被简化...

An erratum was issued for <a href="http://www.jove.com/video/52439">Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)</a>. The disclosures were updated.

An erratum was issued for <a href="http://www.jove.com/video/52439">Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)</a>.

Errata: Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

切体研究是调查的一个新兴领域，它检查携带RNA 1，DNA 2和3的蛋白脂类货物微泡。以前的调查外来体生物学已经导致鉴定外来体的生物流体诸如血液4，尿5，母乳6和唾液7。研究表明，外来体发挥不同细胞途径的作用，远程沉思的体8的不同系统之间的通信。的作用，因为外来体在细胞间通讯玩，假定它们可以打包与疾病状态相关的生物分子靶（蛋白质，RNA和DNA）。 体外 3和动物模型9的研究显示为证实这一假设。在调查的生物标志物发现外来体的内容，有必要制定从生物液体选择性切体隔离的方法，诱导expulsi货物从外来体，和切体生物分子​​的定量。在这项工作中的程度，外来体将被定义为具有直径为约70-100纳米，具有表面标记CD63的结构。 研究人员通常先净化外来体由超速10，然后经过加工裂解液袋使用外体内容。裂解缓冲液的方法的使用需要的孵育时间范围从数分钟到数小时。这个过程可能会潜在危害的外来体货，导致样品降解。例如，通过裂解缓冲液释放到周围的细胞外环境唾液外来体RNA具有的半衰期小于1分钟，使得外来体RNA后裂解的测定缓冲一个特别困难的任务不添加稳定化试剂11。添加各种试剂溶解和稳定的配合作用可以引入该复杂化剂和干扰ANALYSIS的外来体内容。另一种做法可以是用于快速卸外来体内容并安全地保存货物为表征很有帮助。 在这项工作中，我们提出了一个非均匀电场的用法的外来体内容的释放。电场已被公知的携带极化和破坏形成细胞膜脂双层的能力。我们的实验工作探索不均匀环状方波（CSW）的用法用于破坏外来体的微泡结构和​​释放运载的货物。这种方法在几百毫伏范围内使用电压，这意味着大多数生物分子不会中断。我们表明，环状方波的使用能够致动的唾液mRNA的外来体内容释放进入周围环境中的流体。此释放的外来体内容无缝地与可用于量化生物标志物表达水平的电极系统集成12,13，该提出的方法允许快速，灵敏，和裂解缓冲液不含外来体含量分析。 <img alt="图1" src="/files/ftp_upload/52439/52439fig1highres.jpg" /> 图EFIRM工作流1.概述。该EFIRM方法大致分为三个主要阶段所必需的纯化和分析外来体。 这个CSW基于外来体内容释放和分析方法被用在与CD63的特异性磁微珠为外来体隔离结合使用。这些CD63亲和力磁珠允许从唾液样品（和其他生物流体）的外来体的选择性分离。在温育和提取使用磁化珠的外来体，珠粒被迁移到的CSW基于内容发布和实验分析部的电化学传感器系统。 图1给出了工作的概述流动EFIRM方法。

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:157px;">Company</td>
			<td style="width:132px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Helios 16-Channel Reader System with Chip Interface&#160;</td>
			<td style="width:157px;">Genefluidics, USA</td>
			<td style="width:132px;">&#160;RS-1000-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16X Sensor Chip, Bare Gold, pack of 5 chips</td>
			<td style="width:157px;">Genefluidics, USA</td>
			<td>SC1000-16X-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Biotinylated anti-human CD63 Antibody</td>
			<td style="width:157px;">Ancell, USA</td>
			<td style="width:132px;">215-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dynabeads MyOne Streptavidin T1</td>
			<td>Invitrogen, USA</td>
			<td>65601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Neodynium Magnetics ( 1/10&#34; dia. x 1/32&#34; thick)</td>
			<td style="width:157px;">K&#38;J Magnetics, USA</td>
			<td>DH101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Ultrapure Distilled Water</td>
			<td style="width:157px;">Life Technologies, USA</td>
			<td>10977-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Mettler Toldeo 3M KcL Solution</td>
			<td style="width:157px;">Fisher Scientific, USA</td>
			<td>1911512</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Pyrrole</td>
			<td style="width:157px;">Sigma-Aldrich, USA</td>
			<td style="width:132px;">W338605-100g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Anti-Fluorescein-POD, Fab fragments</td>
			<td style="width:157px;">Roche, Germany</td>
			<td>11426346910</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:347px;">3, 3&#8242;, 5, 5&#8242; tetramethylbenzidine substrate (TMB/H2O2, low activity)&#160;</td>
			<td style="width:157px;">Neogen, Usa</td>
			<td style="width:132px;">330175</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Phosphate Buffered Saline Solution</td>
			<td style="width:157px;">Life Technologies, USA</td>
			<td align="right" style="width:132px;">10010023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Casein/PBS</td>
			<td style="width:157px;">Fisher Scientific, USA</td>
			<td>37532</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of exosomal biomarker by electric field-induced release and measurement (efirm)

外来体是起到间通讯中介的作用，小泡结构。这是研究的外来体的内部货物以确定它们是否携带疾病的生物标志物的歧视性的兴趣。用于执行外来体分析，有必要开发用于提取和分析来自靶生物流体的外来体，而不会损坏内部内容的方法。 电场诱导释放，并测量（EFIRM）是用于具体提取外来体从生物流体，卸载他们的货物，并测试其内部的RNA /蛋白质含量的方法。使用抗人类CD63特定抗体的磁性微粒，外来体被首先从生物流体中沉淀。以下提取，低压电环状方波（CSW）被施加到破坏泡囊膜而引起的货物装卸。外来体的含量杂交固定在电极表面，用于QUA的DNA引物或抗体的ntification分子内容。 该EFIRM方法是有利的提取外来体和卸载货物中的分析，而无需裂解缓冲液。该方法是能够执行RNA和蛋白质生物标志物的目标的特异性检测，在外来体中。 EFIRM提取具体地说明在其表面上的标记的外来体，而不是基于大小的技术。 透射电子显微镜（TEM）和测定表明该方法对于外来体捕获和分析的功能。该EFIRM方法用于外来体注射人肺癌细胞H640 9小鼠的分析，以测试它们的外来体信息对11只小鼠接受盐水对照（转染以表达外来体标记人类CD63-GFP的细胞系）。外体生物标志物水平升高（参照基因GAPDH和蛋白质表面标志CD63人-GFP）被发现的H640小鼠注射血清和唾液样本。此外，贝VA和血清样品证明具有线性关系（r = 0.79）。这些结果暗示对唾液外来体的生物标志物用于检测远端疾病的可行性。

珠用TEM的外来体捕获验证 以下通过使用透射电子显微镜（TEM）图像提取协议使用抗人类CD63磁性珠从唾液外来体的分离进行了验证。 TEM显示磁珠与70-100纳米颗粒直接相邻（ 见图3A和3B），与外来体的已知轮廓相一致。观察为磁珠唾液那些没有缀合到它们的抗人CD63抗体先前（ 见图3C和3D）无70-100纳米颗粒。 <p cla...

Watch this Scientific Journal Video about Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM at JoVE.com

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

通过电场诱导释放和测量（EFIRM）检测外体生物标志物

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

detection-exosomal-biomarker-electric-field-induced-release

Research

JoVE Journal

Bioengineering

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

18.3K 次观看.  University of California, Los Angeles. Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves. 

视频: Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

开发自定义的中国仓鼠卵巢宿主细胞蛋白检测使用声膜微粒子技术

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

科研

生物工程

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

总结凝聚力测量组织表面Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.
Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.

We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.

在激光诱导轴突病变张力释放测量评估轴突在Piconewton和毫秒级的分辨率对基材的附着力

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

光学检测<em&gt; E。大肠杆菌</em&gt;由细菌介孔硅生物传感器

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

最大等长收缩力的测量通过透骨骼肌纤维生成

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

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

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

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

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

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

微流体设备中的电场诱导神经前体细胞分化

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

细胞电微环境的有限元素建模

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

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, ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.