Name: a microfluidic chip for the versatile chemical analysis of single cells
Uploaded: 2013-10-15T17:00:00
Description: Watch this Scientific Journal Video about A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells at JoVE.com

作者非常感谢汤姆罗宾逊的手稿，C.Bärtschi和H.奔驰的定制压力控制系统建设的校对。我们还要承认使用洁净室设施第一和光学显微镜中心（LMC），无论是在ETH苏黎世。这项工作是在第七框架计划（ERC开始格兰特，项目编号203428，nμLIPIDs）资助下，由默克雪兰诺公司和欧洲研究委员会（ERC）。

1。 SU-8制作大师准备两个主模具的通道（流体和控制，原理图及尺寸见图2）以下协议，但不同的掩模图案。该过程示于图3a。 <ol><li>开始通过10分钟在180℃下加热4英寸硅晶片装载于旋涂机脱水晶片，并使用下列协议旋涂SU-8 2015： <ol><li>旋转晶片，在100 rpm离心20秒（开放旋涂机的盖子，分配后在该步骤中，盖上盖子分配的SU-8）。 </li><li>旋转晶片，在500...

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微流体技术开辟为单细胞分析的新的和引人入胜的可能性。特别是，可能性陷阱和微流控工具分别固定细胞已经允许在单细胞特性和响应系统的短期和长期的研究。此外，细胞在高频率的微滴，在微型芯片上所产生的封装性，使单个细胞分泌的研究，这不能用传统的流式细胞仪设备来执行。微滴的方法，但是，当有需要的分析协议孵化和洗涤步骤一定的局限性。本方法结合了细胞俘获和隔离的两?...

在过去的许多研究已经揭示了大量的细胞群1-3中的细胞-细胞间的差异，特别是信号处理4，或细胞内的生物分子如蛋白质5,6，代谢物，辅因子和7,8的量。这些非均匀性被认为是对细胞的适应和进化9极其重要，而且在癌症等疾病63-82的产生和治疗中发挥关键作用。因此，在单细胞水平的研究是在生物和药理研究兴趣高的，尤其是当这些研究揭示了不同的细胞反应与生物活性的化学物质，处理后。 近年来，许多分析平台已经开发了促进单个活细胞的分析或细胞含量的化学组成。而荧光激活细胞分选（FACS）是金圣andard对单个活细胞的非常高通量分析，该方法不能被用于细胞内或分泌化合物的定量。微流体平台的出现已经答应为定位，治疗和观察单个细胞的新的分析策略。微流控芯片中的一个里程碑达成与由地震和同事14,15实现灵活的PDMS阀门的整合。这些阀门是有用的，因为它们可以隔离区域上的芯片， 例如分开两种文化16。此外，它们特别适用于单细胞分析，因此有助于减少分析物的稀释问题。这种方法对单细胞分析的力量已经由汉森和他的同事，谁从几百单细胞在17平行分析的基因表达最近展示。 当靶向蛋白质和代谢物的分析是非常困难的，由于缺乏合适的一mplification方法中，大量不同的化合物存在，和它们的化学性质的变化。此外，大多数胞内生物分子预计将存在于几万种18的次序低的拷贝数，因此所用的分析方法必须具有高灵敏度。更强大的测定例如免疫测定和酶联免疫分析（ELISA）是很难集成到微流控装置，因为它们需要几个洗涤和温育步骤，以及表面固定。 由于这些挑战，这并不奇怪，只有几个例子被报道了，其中蛋白质或代谢物进行定量的单细胞水平。例如，关于荧光化合物的分泌的研究已有报道19,20。最近，用ELISA法的实施，提出了从细胞培养分泌（非荧光）蛋白（THP-1细胞）21和单（我的分析mmune）细胞10。靶向细胞内的蛋白质，Shi 等人开发了促进细胞内蛋白质的鉴定通过免疫测定11的装置的信令在肿瘤细胞途径的分析的微流体装置。然而，蛋白质的唯一的相对量进行了测定，并没有酶促扩增被用来增加对低丰度蛋白的信号。 最近，我们能够以单细胞俘获微型装置用荧光测定法8和免疫22（ 图1）相结合。细胞是被动被困在微米级关卡结构，它允许供应和（快速）交换媒介和其他化学试剂无细胞的任何运动。围绕每个陷阱的环形阀使细胞的分离在一个非常小的量（以下简称“微室”）。该阀引入细胞裂解（低渗）缓冲，他后立即启动NCE防止细胞内分子或分泌的分子扩散而去。最重要的是，由于量（625 PL）的小尺寸，避免大量稀释的分子。此外，由于溶解和分析都在芯片的同一位置进行的，不存在损失，由于运输分析物。这里所描述的芯片设计包括8交替的行，7或8微容器的，共计60微容器。腔被致动以行，从而使交叉污染沿一条线被排除。 该平台可以与荧光测定法，以及免疫学测定法（ 图1d）组合使用。对于后者，我们建立了协议的抗体，这是与芯片的生产和装配过程兼容的固定。因此，该平台开辟了道路敏感，可靠和可计量的测定在单细胞水平。到现在为止，我们已经使用该设备为i的分析ntracellular和分泌的酶（通过酶测定法相对定量），细胞内辅助因子，蛋白质和小分子（绝对定量通过终点测定法或ELISA）。在下文中，我们描述了由多层软光刻和抗体的形成图案的协议通过微接触印刷和表面化学的手段的手段芯片制造的过程。此外，芯片的使用和操作的一些例子给出。

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			<td height="21" style="height:21px;width:344px;">REAGENTS:</td>
			<td style="width:284px;"></td>
			<td style="width:103px;"></td>
			<td style="width:153px;"></td>
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			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:344px;">Name of the Reagent</td>
			<td style="width:284px;">Company</td>
			<td style="width:103px;">Catalogue Number</td>
			<td style="width:153px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">SU-8 2015</td>
			<td style="width:284px;">MicroChem Corp. (Netwon, MA)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">1H,1H,2H,2H-Perfluorodecyl-dimethylchloro-silane</td>
			<td style="width:284px;">ABCR (Karlsruhe, Germany)</td>
			<td style="width:103px;">AB103608</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">4-Methylumbelliferyl &#946;-D-N,N&#8242;-diacetylchitobioside hydrate</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">M9763</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Acetone</td>
			<td style="width:284px;">Merck VWR (Darmstadt, Germany)</td>
			<td style="width:103px;">100014</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Avidin</td>
			<td style="width:284px;">AppliChem (Axon Lab AG)</td>
			<td style="width:103px;">A-2568</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">AZ 1518</td>
			<td style="width:284px;">AZ Electronic Materials (Wiesbaden, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">AZ 726 developer</td>
			<td style="width:284px;">AZ Electronic Materials (Wiesbaden, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Bovine serum albumin</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">A-4503</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Bovine serum albumin, biotin</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">A-8549</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Cell dissociation buffer</td>
			<td style="width:284px;">Invitrogen</td>
			<td style="width:103px;">13151-014</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Hexamethyldisilazane (HDMS)</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">40215</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Hydrochloric acid</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:103px;">84422</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Isopropanol</td>
			<td style="width:284px;">Merck VWR (Darmstadt, Germany)</td>
			<td style="width:103px;">109634</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Magnesium chloride hexahydrate</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:103px;">63068</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">MR developer 600</td>
			<td style="width:284px;">Microresist technology GmbH (Berlin, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">PBS</td>
			<td style="width:284px;">Invitrogen</td>
			<td style="width:103px;">10010-031</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">PLL-g-PEG grafted</td>
			<td style="width:284px;">SuSoS, (D&#252;bendorf, Switzerland)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">PLL-g-PEG grafted biotin</td>
			<td style="width:284px;">SuSoS, (D&#252;bendorf, Switzerland)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Potassium chloride</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:103px;">60132</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Protein G, biotin</td>
			<td style="width:284px;">Sigma Fine Chemicals</td>
			<td style="width:103px;">41624</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Silicon wafer</td>
			<td style="width:284px;">Si-Mat (Kaufering, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Sylgard 184 Silicone Elastomer Kit (PDMS)</td>
			<td style="width:284px;">Dow Corning</td>
			<td>39100000</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Tris(hydroxymethyl)-aminomethan</td>
			<td style="width:284px;">Biorad</td>
			<td style="width:103px;">1610716</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Tween 20</td>
			<td style="width:284px;">Biorad</td>
			<td style="width:103px;">1706531</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:344px;">Material Name</td>
			<td style="width:284px;">Company</td>
			<td style="width:103px;">Catalogue Number</td>
			<td style="width:153px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">0.22 &#181;m PES syringe filter</td>
			<td style="width:284px;">TRP</td>
			<td style="width:103px;">99722</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">1/1.5 mm biopsy puncher</td>
			<td style="width:284px;">Miltex, York PA</td>
			<td style="width:103px;">33-31AA/33-31A</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Cell Trics filter 20 &#181;m</td>
			<td style="width:284px;">Partec</td>
			<td style="width:103px;">04-004-2325</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Centrifuge Sigma 3-18K</td>
			<td style="width:284px;">Kuehner</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Hotplate HP 160 III BM</td>
			<td style="width:284px;">Sawatec, Sax, Switzerland</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">MA-6 mask aligner</td>
			<td style="width:284px;">Karl Suess</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Multizoom AZ100M microscope</td>
			<td style="width:284px;">Nikon Corporation</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Photomask</td>
			<td style="width:284px;">Microlitho, Essex, U.K.</td>
			<td style="width:103px;">n.a</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Plasma Cleaner PDC-32G</td>
			<td style="width:284px;">Harrick</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Spin coater Modell WS-400 BZ-6NPP/LITE</td>
			<td style="width:284px;">Laurell</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Spin Modules SM 180 BM</td>
			<td style="width:284px;">Sawatec, Sax, Switzerland</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Step profiler Dektak XT Advanced</td>
			<td style="width:284px;">Bruker</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Syringe pump neMESYS</td>
			<td style="width:284px;">Cetoni</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
	</tbody>
</table>

a microfluidic chip for the versatile chemical analysis of single cells

我们提出了一个微流体装置，使多个单体电池并联定量测定细胞内生物分子。为此目的，将细胞被动被困在一个微容器的中间。在激活时，控制层，所述细胞是从一个小的腔室周围的容积隔离。周围的体积然后可以在不影响分离的细胞进行交换。然而，在该腔室的短的开闭，在该腔室中的溶液可在几百毫秒取代。由于腔室的可逆性，可以将细胞暴露于不同溶液依次以高度可控的方式， 例如 ，用于温育，洗涤，最后，细胞裂解。该密封的微容器使裂解液的潴留，尽量减少和细胞裂解后，控制稀释。自体溶解和分析出现在同一位置，高灵敏度被保持，因为没有进一步ðilution分析物的损失或运输过程中发生。因此，微室的设计使之从单个细胞释放的细胞内分子（阿托摩尔，zeptomoles）非常小的拷贝数的可靠和可重复的分析。此外，许多微容器可以被布置成阵列格式，允许在一次许多细胞的分析，考虑到合适的光学仪器被用于监视。我们已经使用该平台的概念验证研究，分析细胞内蛋白质，酶，辅助因子和相对或绝对量化的方式第二信使。

我们的平台能够分析存在于或由单个细胞产生的多种细胞内的以及所分泌的分子。在这里，我们想呈现不同的实例研究强调了各种可能的分析。我们将举一个例子，对于分泌的酶（ 图5a），以及细胞内的酶（ 图5b）和蛋白质（ 图5c和d）所示 。更多的例子，如辅助因子或小分子，请参阅Eyer 等（2012）和Eyer 等人 （2013）。 <p class="jo...

Watch this Scientific Journal Video about A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells at JoVE.com

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

在这篇文章中，我们提出了一个微流控芯片单细胞分析。它使细胞内的蛋白质，酶，辅因子，以及第二信使通过荧光测定法或免疫测定装置的量化。

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Research

JoVE Journal

Bioengineering

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

微流控装置重新创建一个肿瘤微环境<em在体外</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

科研

生物工程

A Microfluidic-based Hydrodynamic Trap for Single Particles

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 ...

Fluorescence detection methods for microfluidic droplet platforms

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

A Microfluidic Chip for ICPMS Sample Introduction

微流体芯片ICPMS进样

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

活微生物组共培养与人的微工程肠绒毛在肠道上一个芯片微流体装置

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

设计微流体装置用于研究细胞反应在单人或共存化工/电气/剪切应力刺激

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

A Versatile Method of Patterning Proteins and Cells

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...