Name: automated lipid bilayer membrane formation using a polydimethylsiloxane thin film
Uploaded: 2016-07-10T13:00:00
Description: Watch this Scientific Journal Video about Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film at JoVE.com

This work was supported by the Pioneer Research Center Program (NRF-2012-0009575) and National Research Foundation Grants (NRF-2012R1A1B4002413, NRF-2014R1A1A2059341) from the National Research Foundation of Korea. This work was also partially supported by the Inha University Research Grant.

1.溶液的制备<ol><li>缓冲溶液的制备： <ol><li>配制缓冲液中，溶解的1M的KCl（氯化钾）的10mM的Tris-HCl液（Tris-盐酸盐），和1mM EDTA（乙二胺四乙酸）在蒸馏水和调节pH值至8.0。 </li><li>过滤器采用0.20微米的过滤器解决方案。灭菌，高压灭菌，在121℃下的溶液15分钟。 </li></ol></li><li>对于预涂装脂溶液的制备： <ol><li>制定用于预喷漆的脂质溶液，溶解3％DPhPC（1,2- diphytanoyl- SN -glycero -3-磷脂酰胆碱?...

<ol>
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Our BLM formation technique provides a powerful tool for cell membrane and ion channel studies, in contrast to conventional techniques that have limited potential for industrial use. We developed a membrane precursor using a PDMS thin film, and devised a frozen membrane precursor with expedited self-assembly.
As opposed to conventional membrane formation methods with hydrophobic films, where membrane formation only occurs via surface interactions between the film and the lipid solution,20...

人工脂质双层膜，或者黑脂质膜（BLM），为阐明细胞膜和离子通道，机制以及对于理解离子通道和离子/分子之间的相互作用的一个重要工具。1-7虽然膜片钳方法通常被认为是对细胞膜的研究的金标准，它是费力的并且需要对离子通道测量高度熟练的操作人员。8虽然人工重组的脂质双层膜已成为对离子通道的研究替代工具，9,10-它们也与费力相关联流程和具体的专业知识。此外，膜是易受机械扰动。因此，引入到日期脂质双层技术具有有限的实际应用。11 为了增强脂质双层膜，Costello 等人 12，和IDE和柳田的稳健性和寿命<suP&gt; 13已经设计出由水凝胶支持一个独立的脂双层。然而，尽管增强长寿（&lt;24小时），双层稳健性没有得到改善。全度等人 14设计了一种水凝胶包封的膜（HEM）与亲密水凝胶的脂双层的接触，从而增强了寿命（长达数天）。为了进一步增加HEM的寿命，Malmstadt和全度等人创建了水凝胶的脂质通过原位共价缀合（cgHEM）。15结合在这两个系统的水凝胶包封的膜，膜的寿命显着增加（&gt; 10天） 。然而，膜形成系统是不足够坚固，并且在必要时以释放专门知识的使用脂双层的不能被存储或传送。 脂质双分子层平台的开发越来越周围的鲁棒性和类脂膜的寿命已初步旋转。虽然类脂膜的寿命一直是苏最近bstantially提高，其应用被限制由于缺乏输送和耐贮性的。为了克服这些问题，全度妍等人创建了一个可存储的膜系统，并推出了隔膜前体（MP）。16要构建MP，他们准备正癸烷和十六烷的含有3％DPhPC（1,2-diphytanoyl-混合SN -glycero -3-磷脂酰胆碱）来控制脂质溶液，使得其将在〜14℃（室温以下，上述典型的冰箱温度）冻结的冰点。在该实验中，在MP被分布在一个小的孔上的聚四氟乙烯（PTFE）膜，并随后在4℃的冰箱中冷冻。当MP被带到室温，在MP解冻和脂质双层中自动形成的，消除了通常与膜形成相关的专门知识。然而，从MP制成BLM的成功率较低，为〜27％，和膜formation个时间不一致（30分钟至24小时），限制了其实际应用。 在这项研究中，如先前通过柳报道聚二甲基硅氧烷（PDMS）薄膜来代替传统的疏水性薄膜（聚四氟乙烯，聚甲醛，聚苯乙烯）至（a）控制制造时间和（b）增加BLM形成的成功率等 17在这里，膜的形成是通过溶剂萃取，由于PDMS的多孔性容易，并且对于膜形成所需的时间在本研究中被成功控制。在这个系统中，作为脂质溶液吸收到的PDMS薄膜，一致的膜形成时间达到了。此外，膜的寿命延长，由于溶剂的缓慢吸收进入的PDMS薄膜，相加角鲨烯的到脂质溶液的结果。我们进行了光学和电学测量，以验证使用这种技术形成的膜是合适的对于i对渠道的研究。

<table border="0" cellpadding="0" cellspacing="0" width="1448">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Potassium Chloride</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">P9333</td>
			<td style="width:743px;">For buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Tris-hydrochloride</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">1185-53-1</td>
			<td>For buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">60-00-4</td>
			<td>For buffer solution</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:335px;">n-decane</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">44074-U</td>
			<td>For lipid solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Hexadecane</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">544-76-3</td>
			<td>For lipid solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Squalene</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">S3626</td>
			<td>For lipid solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Gramicidin A</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:199px;">11029-61-1</td>
			<td>Membrane protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">1,2-diphytanoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:172px;">Avanti Polar Lipids, Inc.</td>
			<td style="width:199px;">850356</td>
			<td>For membrae formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Sylgard 184a and 184b elastromer kit</td>
			<td style="width:172px;">Dow Corning Asia</td>
			<td style="width:199px;"></td>
			<td>To produce PDMS thin film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">0.2 &#956;m filter</td>
			<td style="width:172px;">Satorius stedim</td>
			<td style="width:199px;">16534----------K</td>
			<td>To filter buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Rotator</td>
			<td style="width:172px;">FinePCR</td>
			<td style="width:199px;">AG</td>
			<td>To dissolve lipid homogeneously</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Autoclave</td>
			<td style="width:172px;">Biofree</td>
			<td style="width:199px;">BF-60AC</td>
			<td>To sterilize buffer solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Spin coater</td>
			<td style="width:172px;">Shinu Mst</td>
			<td style="width:199px;">SP-60P</td>
			<td>To spread PDMS prepolymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Vaccum dessiccator</td>
			<td style="width:172px;">Welch</td>
			<td style="width:199px;">2042-22</td>
			<td>To remove air bubble in PDMS prepolymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">500 &#956;m&#160; punch</td>
			<td style="width:172px;">Harris Uni-Core</td>
			<td style="width:199px;">0.5</td>
			<td>To create an aperture on the PDMS thin film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">CNC machine</td>
			<td style="width:172px;">SME trading</td>
			<td style="width:199px;">SME 2518</td>
			<td>To fabricate membrane formation chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Halogen fiber optic illuminator</td>
			<td style="width:172px;">Motic</td>
			<td style="width:199px;">MLC-150C</td>
			<td>To illuminate the aperture of PDMS thin film for optical observation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Digital microscope</td>
			<td style="width:172px;">Digital blue</td>
			<td style="width:199px;">QX-5</td>
			<td>To optically observe lipid bilayer membrane formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Electrode</td>
			<td style="width:172px;">A-M Systems</td>
			<td style="width:199px;"></td>
			<td>To electrically observe membrane formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Microelectrode amplifier (Axopatch amplifier)</td>
			<td style="width:172px;">Axon Instruments</td>
			<td>Axopatch 200B Amplifier</td>
			<td>To measure capacitance of the membrane (described as microelectrode amplifier in the manuscript)</td>
		</tr>
	</tbody>
</table>

automated lipid bilayer membrane formation using a polydimethylsiloxane thin film

人工脂质双层，或黑脂质膜（BLM）是用于研究离子通道和蛋白质的相互作用，以及用于生物传感器的应用的有力工具。然而，传统的BLM形成技术具有若干缺点，它们经常需要特定的专门技术和费力的过程。特别是，传统的类脂膜从低形成的成功率和不一致的膜形成时间受到影响。在这里，我们通过替换传统使用的薄膜（聚四氟乙烯，聚甲醛，聚苯乙烯），聚二甲基硅氧烷（PDMS）展示与控制稀疏的时间和提高BLM形成率可存储和运输的BLM形成系统。在这个实验中，使用如PDMS薄膜的多孔构造的聚合物。此外，相对于具有低粘度的常规使用的溶剂，使用角鲨烯的通过溶剂吸收慢允许受控稀疏时间由PDMS，延长膜的寿命。在广告DITION，通过使用角鲨烯和十六烷的混合物中，脂质溶液的凝固点增加（〜16℃），此外，膜前驱体被生产，可以无限期储存和容易运输。这些膜的前体具有降低&lt;1小时的BLM形成时间和达到〜80％BLM形成率。此外，与短杆菌肽A离子通道的实验证实了膜系统的可行性。

MPES解决结构优化 脂质和溶剂的不同组合物进行测试，以从MPES成功重组脂质双层膜。在MP系统用正癸烷和十六烷含3％DPhPC 14的混合物表现出的膜形成（〜27％）的低成功率。另外，作为与PDMS膜连续地抽出脂质溶液，有必要优化溶剂组合物以维持完整的脂质双层膜。因此，角鲨烯，其在20℃具有12厘泊的粘度-C 18代替正-癸烷，其...

Watch this Scientific Journal Video about Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film at JoVE.com

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

我们展示了一个可存储，运输的脂质双分子层形成系统。的脂质双层膜可以在1小时内超过80％的成功率，当冷冻膜前体达到环境温度下形成。该系统将减少与离子通道相关的繁琐流程和专业知识。

自动化脂双层膜的形成使用聚二甲基硅氧烷薄膜

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Research

JoVE Journal

Bioengineering

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

基于几丁聚糖，激光激发薄膜手术胶粘剂，“SurgiLux的制备及示范

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

科研

生物工程

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

创建瞬态细胞膜毛孔，使用标准的喷墨打印机

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

复乳代使用聚二甲基硅氧烷（PDMS）同轴流量聚焦设备

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

人工脂质纳米管网作为内质神经的底部模型的自发形成与重排

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

热、压水固化重组蜘蛛丝的材料形成

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...