作者感谢IZON科技有限公司的支持。这项工作是由欧洲委员会研究（PCIG11-GA-2012-321836 Nano4Bio）的支持。

1.用吐温20（PBST）缓冲使磷酸盐缓冲液<ol><li>溶解1的PBS片剂（0.01M磷酸盐缓冲液，0.0027 1M氢氯化物，0.137氯化钠，pH值7.4）在200毫升的去离子水（18.2兆欧厘米）。 </li><li>加入100微升（0.05（V / V）％）吐温20至200毫升缓冲液溶液作为表面活性剂。 </li></ol> 2.准备羧基聚苯乙烯颗粒标准<ol><li>涡旋超声2分钟前30秒的校准颗粒在80瓦到创建粒子的单分散性。 </li><li>稀释校准颗粒在100 1至在PBST缓冲液和涡旋1×10 10个粒子/ ml的浓度为30秒。 </li></ol> 3.准备链亲和素包覆颗粒<ol><li>涡旋超声2分钟前30秒的颗粒在80瓦的单分散性保证。 </li><li>稀释链霉包衣颗粒1 100在PBST缓冲到交流hieve的1×10 9个粒子/ ml，30秒所得浓度和旋涡。 注:一个典型的样本量为200微升。例如，如果调查不同的DNA的浓度制备1ml适当稀释的链霉亲涂覆颗粒。 </li></ol> 4.寡核苷酸的制备<ol><li>重构用去离子水的寡核苷酸到所得100μM的浓度。 </li></ol> 5.加成捕获探针（CP）的DNA的链亲和素包被的颗粒<ol><li>之前DNA结合，涡流链霉包衣颗粒（200微升样品体积）30秒，随后通过一个2分钟超声处理在80瓦。 </li><li>基于由供应商（4352皮摩尔/毫克）所提供的结合能力，加DNA的适当的浓度在颗粒为10，20，30，40，47，95，140，和210纳米的DNA得到的浓度。 </li><li>涡旋10秒并置于样品上在室温下在旋转轮30分钟以允许将DNA通过链霉抗生物素相互作用结合到粒子表面。 </li><li>一旦捕获的DNA已被添加，并与链霉亲包衣颗粒孵育，通过将试样放在一个磁架30分钟除去在经由磁分离溶液中的过量的DNA。 </li><li>除去上清液，注意不要扰乱最接近磁铁粒子的新形成的群集，并用相同体积的新的PBST缓冲液的更换。 </li></ol> 6.杂交的互补DNA到CP颗粒<ol><li>添加所需的靶DNA（超过500纳米）的量，以确保最大可能的靶结合为止。 </li><li>涡旋样品10秒和地点上在室温下在旋转轮30分钟。 </li><li>一旦杂交完成后，通过将样品上的磁架30分钟除去通过磁分离过量的靶DNA。 </li><li>除去上清液，注意不要扰乱最接近磁铁粒子的新形成的群集，并用相同体积的新的PBST缓冲液的更换。 </li><li>重复步骤重复样本6.1至6.4，并放置这些样品，在旋转轮在室温下16小时，以研究DNA杂交时间。 </li></ol> 7. TRPS设置<ol><li>插入仪器到计算机系统中以代替软件。 </li><li>校准用卡尺的初始拉伸。 <ol><li>测量两个平行夹爪的外侧之间的距离。 </li><li>输入通过在"仪器设置"选项卡中输入在"拉伸"字段拉伸，然后点击"校准舒展"的标签下的软件。 </li></ol></li><li>横向适合分析适当大小的聚氨酯纳米孔膜上朝上纳米孔ID号钳口。然后，伸展钳口用于使用分析所需的拉伸在仪器的一侧伸展调节手柄。伸展43和48毫米的下颌。 注意:使校准微粒封锁是至少0.3 nA的尺寸是一起施加电压所确定的拉伸的精确值。拉伸已经输入到软件在步骤7.2和作为钳口被拉伸会自动调整。 </li><li>放置80微升PBST缓冲液中的下部流体细胞，纳米孔的下方，以确保不存在本气泡可能影响测量。如果有气泡看出，取出并更换缓冲。 </li><li>点击上方的流体细胞到位，并把40微升缓冲进去，从而也确保没有泡沫存在。如果气泡存在于上部流体细胞，通过更换液体移除。 </li><li>当可再现的基线电流已从用缓冲液更换上部流体细胞达到，通过点击"ST添加40微升样品到上部流体细胞和测量的"中的"数据采集"软件屏幕上标签的艺术。 注:数据采集完成后，在50千赫的0.05呐封锁幅度下限的频率，虽然可以使用通过"分析数据"选项卡中的软件进行更改（在"分析设置"和"电阻式封锁"） 。 </li><li>放置一个法拉第笼在流体电池系统的顶部，以减少对测量电背景噪声。 </li><li>使用一个可变压力模块（VPM）来的压力或真空施加到样品。 <ol><li>施加的外部压力，喷嘴连接到该上部流体细胞，然后旋转压力臂和卡入到位（取决于是否将被施加正压力（PRE）或真空（VAC））。 </li><li>在使用位于VPM顶部的压力级旋钮&#39;厘米&#39;或&#39;毫米&#39;刻度施加压力。按下旋钮，适用于"厘米"的规模压力，向上拉Ťo在"毫米"规模施加压力。 </li></ol></li></ol> 8.准备的样品分析TRPS <ol><li>涡旋样品在TRPS分析前80瓦特30秒，超声2分钟。 </li></ol> 9.校准纳米孔的泽塔分析<ol><li>将40微升校准颗粒（1×10粒/ ml）倒入上层液体电池后，完成TRPS测量（设置为在第7）在3施加的电压。 " - "按钮上的电压规模在"仪器设置"中的软件选项卡中点击"+"和改变电压。 </li><li>检查3电压回到约140，110背景电流和80 nA的。保证在中等电压的校准微粒产生至少0.3的平均阻滞大小nA的。 </li><li>施加压力，使所述校准微粒的平均半峰全宽（FWHM）的持续时间至少是0.15毫秒。为此使用连接到可变压力模块的压力臂手动。选择压力（PRE）或真空（VAC），直到它卡在期望的位置，并且在步骤7.8.2所适用相应以下设置指示所述臂旋转。一旦这些条件已经实现，通过点击"数据采集"选项卡中的软件&#39;开始&#39;开始运行。 </li><li>当至少有500个颗粒进行了测量（见"粒子计数"在软件屏幕在测量过程中底部），运行已超过30秒，由"数据采集"标签按下"停止"完成运行（参见"运行时间&#39;也朝向屏幕的底部）。 </li><li>通过描述每一种新的纳米孔引入时间或分析每一个新的一天完成步骤9.1-9.4完成校准运行校准系统。 </li></ol> 10.运行样品<ol><li>运行在最高的样品或第二高电压作为确保类似的（±10 NA）的校准样品，如果不相同，基线电流。 <ol><li>一旦适当的基准电流来实现，用40微升样品的替换在上部流体单元中的电解质。当引入的样品，封锁将看到的信号路径。开始通过点击"开始"中的"数据采集"选项卡中运行样品并记录最低500粒子（检查&#39;粒子计数"信号走线下位于），并确保运行时间最少30秒（见"运行时代"也位于信号迹线下方）。 </li><li>要完成测量，点击"停止"中的"数据采集卡"并保存数据文件。 </li></ol></li><li>要保存文件，输入如下格式的文件信息; &#39;调查&#39;是文件将保存在该文件夹，"纳米孔ID&#39;正在被使用，&#39;部分＃&#39;我毛孔的序列号š孔的类型（ 即 ，NP150 / NP200），&#39;样品ID&#39;是样品的名字，"校准或样品"的信息是否是一个校准或测量样品，"稀释"，如果样品稀释被使用（键入100如果样品稀释100倍），"压力"是所施加的压力到样品（以厘米 - 见第7.8节），&#39;电解质ID"是的缓冲样品中由姓名，和&#39;注意事项&#39;是有关样本或运行任何个人笔记。 </li><li>每个样品运行之间，通过将40微升的PBST缓冲液到上部流体细胞几次并施加各种压力（在-10通常，-5厘米（真空），以及5和10厘米（正压力）），直到洗系统没有更多的阻断事件都存在，确保有残留在系统中没有残留的粒子和样品之间，因此没有交叉污染。在这种洗涤步骤每个重复样品之间完成一式三份样品运行身份运行WELl由于不同样本之间。 </li></ol>

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<html>为ζ电势用于计算由Arjmandi 等人 21的工作有关的校准基础的方法。因为它们穿过纳米孔被测量作为所施加电压的函数，使用平均电场和粒子速度定期锥形孔的整体上颗粒的易位持续时间。的电泳迁移率是1 / T（其中T是封锁持续时间）相对于电压的导数，乘以感应区长度的平方， 湖在通过传感区域的多个基准点的平均速度测量以允许最小的误差在使用这种方法计算ζ电位。...

官能化的纳米颗粒变得如在医疗和环境领域的生物传感器日益流行。的能力，以改变纳米颗粒的表面化学，用DNA，例如，被证明是用于靶向药物递送系统1和监测DNA-蛋白质相互作用2-4是有用的。被利用在生物测定一个越来越普遍的纳米颗粒性质和在治疗剂的递送是超顺磁性5。超顺磁性粒子（SPP的）是在确定和从复杂混合物中去除特定分析物是非常有用的，可以用简单的使用单个磁体的这样做。一旦移除，所述分析物结合的颗粒可以被表征和分析适合目的。 用于纳米颗粒的检测和表征以前的方法包括光学技术，诸如动态光散射（DLS），或称为光子相关光谱法。虽然一喜GH可以通过技术，DLS被限定于一个平均为基础的技术，并没有加入专业软件分析多峰样品时，较大的颗粒将产生一个更主导信号，留下一些较小的颗粒完全被忽视6,7。粒子按颗粒表征技术，因此更利于分析和纳米功能化纳米粒子系统。 基于RPS技术基于周围施加电场到样品，并通过合成或生物纳米孔监测粒子的输送机构。根据RPS相对近期的纳米检测与表征技术是可调电阻脉冲感应（TRPS）8-16。 TRPS是由弹性，可调谐孔隙膜分开的两个电极系统。的可调孔径的方法允许在各种形状17和大小的分析物通过其反待测量通过毛孔口的机制。可调孔隙先前已经用于生产比较的结果，其他技术如透射电子光谱法（TEM）10检测的小颗粒（70-95纳米直径）。当施加电场时，离子电流被观察到并作为颗粒/分子穿过孔，它们暂时阻止孔径，导致在当前的减少，可以被定义为一个"封锁事件&#39;。每个封锁事件是代表单个颗粒，使得样本内的每个颗粒可以表征单独基于所述封锁幅度，Δ <img alt="式（1）" src="/files/ftp_upload/54577/54577eq1.jpg" /> 和全宽度半最大，FWHM，以及其他封锁性能。判断单个颗粒，因为他们通过纳米孔为多峰的样品有利的，因为TRPS可以成功地和有效区分的范围内的颗粒尺寸阿蒙GST一个样本。可调电阻脉冲感测完成大小10，同时ζ电位12,18和浓度15测量在单次运行，因此，仍可以区分的类似的样品，如果不通过表面电荷19相同的大小;在施胶替代技术的优势。 ζ电位是指在剪切20的平面上的静电势，并作为它们穿过的孔19从粒子速度计算。单个颗粒的ζ电位测量值从而给出洞察易位机制和在溶液中的纳米颗粒系统中，纳米颗粒测定设计未来的有价值的信息用于一系列应用的行为。粒子通过粒子这种性质的分析，还允许之间的样本人群中蔓延，zeta电位值的分布加以探讨，使邻更多信息要达到n个反应动力学（单链到双链DNA，例如）和颗粒稳定性在溶液中。 在这里，我们描述了检测和表征未修饰和DNA修饰SPP表面的技术。本文描述的协议是适用于一系列无机和生物纳米颗粒，但我们证明用DNA修饰表面的方法，由于其广泛的应用。该技术允许用户在纳米颗粒表面的单链和双链DNA靶区分基础上，通过一个孔体系粒子易位速度和因此它们的ζ电势。

<table border="0" cellpadding="0" cellspacing="0" width="602">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate buffered Saline (PBS)</td>
			<td style="width:100px;">Sigma Aldrich, UK</td>
			<td style="width:119px;">P4417</td>
			<td style="width:167px;">1 tablet dissolved in 200 mL deionised water to make buffer solution.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween-20</td>
			<td style="width:100px;">Sigma Aldrich, UK</td>
			<td style="width:119px;">P1379</td>
			<td>0.05% (v/v) in PBS buffer as a surfactant</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Carboxyl polystyrene nanoparticles</td>
			<td style="width:100px;">Bangs Laboratories, US</td>
			<td style="width:119px;">CPC200</td>
			<td>Nominal diamter of 220 nm, raw concentration of 1E12 particles/mL, specific surface charge of 86 &#181;eq/g (equivalent to a surface charge density of 3.2E19 C/nm^2.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Streptavidin coated nanoparticles</td>
			<td style="width:100px;">Ademtech, France</td>
			<td style="width:119px;">3121</td>
			<td>Batch had binding capacity of 4352 pmol/mg (188 nM theoretical DNA binding capacity) at a raw concentration of 1.1E11 particles/mL.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated oligonucleotides</td>
			<td style="width:100px;">Sigma Aldrich, UK</td>
			<td style="width:119px;">VC00001</td>
			<td>Supplier spec: Reverse Phase 1 purification (0.05 Scale); Biotin modification at 3&#39; end; Lyophilised powders reconstituted to 100 &#181;M using deionised water, and diluted as required. Sequences: CP 5&#39;ATGGTTAAACCTCAC 
			TACGCGTGGC[Btn]3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard olignonucleotides</td>
			<td style="width:100px;">Sigma Aldrich, UK</td>
			<td style="width:119px;">VC00001</td>
			<td>Supplier spec: Reverse Phase 1 purification (0.05 Scale); Lyophilised powders reconstituted to 100 &#181;M using deionised water, and diluted as required. Sequences of DNA targets: Fully complementary - 5&#39;GCCACGCGTAGTGAGGTTTAACCAT3&#39;, Middle binding - 5&#39;GTAGTGAGGT3&#39;, End binding - 5&#39;GTTTAACCAT3&#39;, Partially complementary overhanging - 5&#39;GTGAGGTTTAACCAT 
			TTTTTTTTTTTTTTT3&#39;.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Izon qNano</td>
			<td style="width:100px;">Izon Science, NZ</td>
			<td style="width:119px;"></td>
			<td>Inherent pressure on system of 47 Pa,</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Izon Variable Pressure Module (VPM)</td>
			<td style="width:100px;">Izon Science, NZ</td>
			<td style="width:119px;"></td>
			<td>Each &#39;cm&#39; of pressure is equivalent to approximately 1000 Pa.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polyurethane nanopore membranes</td>
			<td style="width:100px;">Izon Science, NZ</td>
			<td style="width:119px;">NP150</td>
			<td>Analyte size range 60-480 nm, pore diameter of calculated to be 799 nm at a 45 mm stretch.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Magrack 6</td>
			<td style="width:100px;">GE Healthcare, UK</td>
			<td style="width:119px;">28-9489-64</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sonic Bath</td>
			<td style="width:100px;">Fisher Scientific, UK</td>
			<td style="width:119px;">10692353</td>
			<td>80 Watts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortexer</td>
			<td style="width:100px;">IKA, Germany</td>
			<td>0003365000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rotary Wheel&#160;</td>
			<td style="width:100px;">Labnet International, US</td>
			<td style="width:119px;">H5500-230 V</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of zeta potential via nanoparticle translocation velocities through a tunable nanopore: using dna-modified particles as an example

纳米孔技术，统称为电阻脉冲传感器（RPS），被用来检测，量化和表征的蛋白质，分子和纳米颗粒。可调电阻脉冲感测（TRPS）是一个相对较新的适应RPS并入，可以实时地改变的可调孔径。这里，我们使用TRPS监测DNA改性的纳米粒子的易位倍，因为它们穿过可调孔膜作为DNA浓度和结构的一个功能（ 即 ，单链到双链DNA）。 TRPS是基于两个的Ag / AgCl电极，通过它建立在所施加的电场产生稳定的离子电流的弹性体孔膜分离。与各种基于光学颗粒表征技术，TRPS可以表征单个颗粒之间的样本人口，允许能够轻松进行分析多式联运样品。这里，我们证明zeta电位测量通过已知的标准粒子易位速度和应用这些样品的分析物易位倍，从而导致测量这些分析物的Zeta电位。 以及获得的平均ζ电位的值，将样品全部采用的粒子由粒子立体显示通过样品人口分布的给定样品的详细信息，例如测量的。这样，这种方法演示了医疗和环境等领域的传感应用中的潜力。

<img alt="图1" src="/files/ftp_upload/54577/54577fig1.jpg" /> 图1. 磁性纯化和TRPS测量过程的示意图。 A）的开始与含有样品过量，未结合的捕获探针的DNA。B）的样本的磁性纯化的实施例的TRP测定例ⅰ）粒子穿过纳米孔和ii）从在孔粒子暂时阻塞离子封锁事件产生使暂时降低在目前的;从被用来计算粒子易位速度的信息。 <a href="https://www.jove.com/fi...

Watch this Scientific Journal Video about Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example at JoVE.com

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

在这里，我们使用集成到一个电阻脉冲感测技术经由粒子易位速度的测量，这可以被用来确定各个纳米粒子的ζ电位来表征纳米颗粒表面化学的聚氨酯可调谐纳米孔。

通过通过纳米孔可调纳米易位流速Zeta电位测定:利用DNA修饰颗粒为例

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

determination-zeta-potential-via-nanoparticle-translocation

Research

JoVE Journal

Bioengineering

12.1K 次观看.  Loughborough University. 在这里，我们使用集成到一个电阻脉冲感测技术经由粒子易位速度的测量，这可以被用来确定各个纳米粒子的ζ电位来表征纳米颗粒表面化学的聚氨酯可调谐纳米孔。 

视频: Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

超声造影有针对性的治疗胶质瘤的小鼠通过药物轴承纳米粒子交付和微血管消融

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

科研

生物工程

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

一个单粒子为基础的微流控液力陷阱

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

定量和大小分析外囊泡的使用可调电阻脉冲检测

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

细菌表面电位测量用开尔文探针力显微镜

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

非透明的支架成像细胞活性 - 使用新型针织钛种植体为例

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the light distortion during the propagation within the fiber. Our methodology is based on digital optical phase conjugation employing only a single spatial light modulator, where the optical wavefront is individually modulated at different regions of the modulator, one region per light signal. Digital optical phase conjugation approaches are considered to be faster than other wavefront shaping approaches, where (for example) a complete determination of the wave propagation behavior of the fiber is performed. In contrast, the presented approach is time-efficient since it only requires one calibration per light signal. The proposed method is potentially appropriate for spatial division multiplexing in communications engineering. Further application fields are endoscopic light delivery in biophotonics, especially in optogenetics, where single cells in biological tissue have to be selectively illuminated with high spatial and temporal resolution.

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

通过光纤使用波前整形多个信号的传输

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.