我们感谢迈克Vahey从弗莱彻实验室在加州大学伯克利分校进行的microjetting参数的建议。这项工作是由赞助国立卫生研究院授予DP2 HL117748-01。

1。无限制造商会<ol><li>设计的无穷室使用电脑辅助设计（CAD）软件（命名为它的形状），然后保存文件，这样它是用激光切割机兼容。在由中心到中心的距离的0.15英寸在透明的丙烯酸用激光切割器切割腔室从1/8- 3/16创建这种形状，直径0.183分开两个圆。无穷大的形状有利于液滴界面双层的形成和稳定。 </li><li>通过丙烯酸系室的边缘钻一个1/16英寸的孔到无穷大形井。重复的相反侧。切离为0.2mm的丙烯酸板用剪刀或激光切割器，以充当一个底部至孔中的小矩形。 </li><li>涂上薄薄但完整层的快干粘合剂，以0.2mm的丙烯酸类的一侧，并且它粘附到所述腔室的底部。持紧密代替0.2毫米丙烯酸对腔室的底部和分配胶在界面以允许胶以形成密封，但避免覆盖可视面积。一定要对齐丙烯酸，使其边缘与室壁的边缘齐平，它完全覆盖了无穷室缺口。这将允许足够的射流穿透和防止井漏。 </li><li>切成两小块天然橡胶的覆盖钻孔。孔周围施加快干粘合剂。将橡胶过孔，然后按上所有区域有一对钳子固定的。重复这两个钻孔。确保所有胶合连接完成密封，以防止任何泄漏。 </li><li>在针使用23 G，1，戳在腔室的两侧中的天然橡胶的孔，以便插入在压电喷墨头的。 </li></ol> 2。实验准备在氯仿在-20℃下冷冻储存股票脂质的解决方案。在这项研究中，无论是1,2 - diphytano基-sn-甘油基-3 -磷酸胆碱（DPhPC）或1,2 -二油酰-sn-甘油基-3 -磷酸胆碱（DOPC）中的使用。 2毫升25毫克/毫升脂质溶液通常制备在本协议中并储存在-20℃下时，将持续两个月<ol><li>重悬脂中的正癸烷中，首先从一个股票的小瓶将其传送到一个小的玻璃瓶中，并在氩气或氮气轻轻擦干。握住瓶子成一角度而干燥，从而暴露更多的表面积与气体，从而使脂质干燥更快。 </li><li>只略微拧在瓶子的盖子，让干燥的溶液中，以在真空下坐于干燥器中1-2小时。然后加入正癸烷再溶解脂质在25毫克/毫升。涡流脂质溶液短暂超声处理它在水浴超声15分钟。存储在正癸烷的重组脂质在-20℃下</li><li>准备一只股票的蔗糖溶液。在1.5毫升微量离心管中，加入900微升300mM的蔗糖和100微升的1％甲基纤维素（MC）。甲基纤维素中加入，以使溶液，这有助于喷射的粘度。可选步骤:添加一个可选的1微升的深色食物染料或荧光珠，借出更多的对比度或荧光成像分别。该解决方案是一个300毫渗量的蔗糖溶液，设计用于蜂窝渗透压和microjetting期间提供的对比。 </li><li>绘制溶液倒入一次性1毫升塑料注射器。握住注射器针尖朝上，反复轻弹轴驱逐向着尖端任何气泡，并推动柱塞弹出被困空中。一定要撤离所有空气，然后再继续注射器，因为它会与适当的压电收缩负责喷射干扰。 </li><li>在注射器的端部安装一个0.22微米的过滤器。以防止空气被截留在过滤器中，垂直握住注射器和推动柱塞，直到液滴的顶端的上方形成。注意:33毫米直径的注射器过滤器单元被发现工作情况最好，但也可用于替代过滤小至3毫米直径的注射器过滤器，以减少死体积。 </li><li>拧开喷墨的阴路厄接头，并牢固地压在适当位置在过滤器的端部。同样，喷射流体，以防止截留的空气。旋入喷墨的顶部。流体应行进到喷墨的前端之后它完全附着。 </li></ol> 3。准备好装备<ol><li>使用v型钳，安装在显微镜载物台的注射器组件。从喷墨连接导线到喷墨控制器。注:自定义阶段是专为这个协议，而舞台设计可以由用户来决定，它有注射器和样品架的XY控制的独立XYZ控制是至关重要的。 </li><li>确定放大率和必要的透镜组合来实现期望的成像。在这里，一个10倍的目标和10X目镜使用。 </li&gt; <li>使用高速摄影机（≥1000帧），以形象化的喷射和囊泡的生成。在此之前成像，进行必要的摄像机标定。利用相机软件中基于图像的自动触发启动图像捕捉。 </li><li>安装无穷室到显微镜载物台。通过录音到位的舞台上固定室。 </li><li>小心地将喷墨针尖刺破用在天然橡胶的孔（参见图1b）。要做到这一点，把喷墨靠近腔和调整由眼定位，然后通过显微镜镜头进行更为精确的调整。步步为营，因为粗动作可能会损坏喷墨一角。 </li><li>一旦喷墨对齐，备份注射器组件远离该腔室，以防止装在井的过程中喷墨的任何损坏。肯定的是，喷墨的运动是单向的，使得它保持与膜中的孔对准。 </li><li>轻轻按下plun注射器组件的GER直到小液滴形式的喷墨喷嘴。这将提供一些初始背压。 </li><li>输入喷射参数。对于双极性梯形波，请使用以下参数:20 kHz脉冲频率，3微秒的上升时间，35微秒的脉冲持续时间，3微秒的下降时间，65 V外加电压（脉冲幅度），并触发每100喷气脉冲（脉冲数） 。然而，所施加的电压（脉冲幅度）和脉冲数（每个触发喷射脉冲）可以变化，以控制泡囊的尺寸。 </li></ol> 4。囊泡生成<ol><li>添加悬浮于正癸烷的无穷室25-30微升脂质溶液，覆盖两孔的全部表面上。 </li><li>加25μl的葡萄糖（同渗容摩为蔗糖溶液）的一个孔中，移液缓慢而平稳的最外边缘。当沉积，减少葡萄糖的形成应​​该的，因为血糖和血脂的解决方案不要混用。添加另一个251; l葡萄糖的相对良好，这将使得再次下降，并在5-10分钟，形成在腔室中部的脂双层膜。 </li><li>插入喷墨穿过天然橡胶，并小心地引导它向着液滴界面双层。接近双层慢慢地，随着引进喷墨将取代量和断裂能的双层。 </li><li>当喷墨内〜200微米，适用于喷射与步骤3.8中所述的设置。从双层的距离可以变化，主要取决于电压和脉冲数，除其他参数。本协议建议缓慢增加设置（​​电压和脉冲数），并观察双层变形。 </li></ol> 5。清洁设备<ol><li>从显微镜载物台取下注射器组装，并处理1毫升的塑料注射器和过滤器。 </li><li>要清洁喷墨，浸渍尖端的solutio吸，以下列解决方案ñ7-10x的各:70％乙醇，2％Neutrad在温水中，70％的乙醇和双蒸2 O溶液如果喷墨不会对吸液管牢固地配合，切枪头，以形成一个变压器。 </li><li>干燥室与组织。将无穷室在250毫升烧杯中，用2％体积/体积Neutrad在温水中，并超声处理5-10分钟。超声处理后，彻底干燥下的压缩空气的水井。任何水分的井可以妥协的脂双层膜的稳定性，因此它也被建议该室被置于烘箱中在60℃下持续15分钟。 </li></ol>

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没有利益冲突的声明。

许多技术已被开发为小泡的产生，包括electroformation，乳液和微滴产生14〜16。然而，新的实验技术是必要的，以允许生物系统的不断增长的相似度来生活系统的设计。特别是微流控方法都提供控制水平的增加管膜unilamellarity，尺寸的单分散性和内部内容17,18，使泡囊模型更接近生物学。此外，使用微流体喷射特性和实验表明定向膜蛋白的有效结合，膜的不对称性，以及封装3,13。...

<html>它已成为越来越明显，细胞生物学是涉及整合我们的理解从分子到细胞的多尺度问题。因此，知道分子正是如何独立工作并不足以理解复杂的细胞行为。这部分是由于多组分系统的涌现行为的存在，作为例证的肌动蛋白网络的互动与脂质双层囊泡4，有丝分裂纺锤体组装在非洲爪蟾的重建中提取5，和细菌细胞分裂机械6空间动态。补充解剖生命系统的分子过程的还原论的方法的一个办法是采取重构的细胞行为使用一组生物成分最小的相反的做法。这种方法的一个关键部分涉及生物分子在一个密闭容积可靠的封装，细胞的一个关键特征。 e_content"&gt;几种策略，用于封装的生物分子为研究仿生系统存在，最生物学相关的系统是脂质双层膜，它模仿了细胞的质膜上施加的生化和物理约束。巨单层囊泡（GUVs）由electroformation 7组，最广泛使用的技术GUV代14之一，通常具有差的包封收率，由于其不兼容的高盐缓冲液8。Electroformation也需要大体积样品（&gt; 100微升），这可能是一个问题，用纯化的蛋白加工和低效地结合由于紧密间隔的脂质层之间扩散的难度大的分子。已经开发了几种微流体方法，用于产生脂质囊泡的双乳剂的方法，它通过多层水 - 油 - 水之间的两个接口传递部件（W / O型/ W）时，依赖于一个VO的蒸发latile溶剂驱动脂质双分子层形成9。他人已经使用产生的脂质双层囊泡10或在两个独立的步骤11的连续流微流体装配线。我们已经开发了基于快速应用流体脉冲对液滴的界面双层12生产控制规模，组成和封装GUVs的替代技术。我们的做法，被称为微流控喷射，提供了从现有几个小泡生成技术的综合优势，为创建功能生物分子系统研究各种生物问题的方法。

<table><tbody><tr><td>Piezoelectric Inkjet</td><td>MicroFab Technologies</td><td>MJ-AL-01-xxx</td><td>xxx denotes orifice diameter in microns</td></tr><tr><td>Jet Drive III Controller</td><td>MicroFab Technologies</td><td>CT-M3-02</td><td></td></tr><tr><td>High-speed camera</td><td>Vision Research</td><td>MiroEX2</td><td></td></tr><tr><td>DPhPC lipid in chloroform</td><td>Avanti</td><td>850356C</td><td>Ordered in small aliquots in vials</td></tr><tr><td>33 mm PVDF filters, 0.2 &amp;micro;m</td><td>Fisher Scientific</td><td>SLGV033RS</td><td></td></tr><tr><td>1 ml Syringes</td><td>Fisher Scientific</td><td>14823434</td><td></td></tr><tr><td>n-Decane</td><td>Acros Organics</td><td>111871000</td><td></td></tr><tr><td>Glucose</td><td>Acros Organics</td><td>410950010</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903-1KG</td><td></td></tr><tr><td>Methylcellulose</td><td>Fisher Scientific</td><td>NC9084958</td><td></td></tr><tr><td>1/8 in Acrylic</td><td>McMaster Carr</td><td>8560K239</td><td>CAD designs for the infinity-shaped chamber are available upon request</td></tr><tr><td>0.2 mm Acrylic</td><td>Astra Products</td><td>Clarex clear 001</td><td></td></tr><tr><td>Acrylic Cement</td><td>TAP Plastics</td><td>10693</td><td></td></tr><tr><td>Loctite 495 Superglue</td><td>Fisher Scientific</td><td>NC9011323</td><td></td></tr><tr><td>Loctite 3494 UV Strengthening Adhesive</td><td>Strobels Supply</td><td>30765</td><td></td></tr><tr><td>Natural rubber</td><td>McMaster Carr</td><td>85995K14</td><td></td></tr><tr><td>Custom stage</td><td>homemade</td><td>N/A</td><td>CAD designs are available upon request</td></tr></tbody></table>

lipid bilayer vesicle generation using microfluidic jetting

自下而上的合成生物学提出了一种新的方法来调查和重构生化系统和潜在的，最小的生物。这一新兴领域从事工程师，化学家，​​生物学家和物理学家来设计和组装基本生物成分为复杂的，功能从下往上的系统。这种自下而上的系统可能导致人造细胞的基本生物学查询及创新疗法1,2的发展。巨型单层囊泡（GUVs）可以作为一个模型平台合成生物学，由于其细胞样膜的结构和尺寸。微流喷射，或microjetting，是一种技术，它允许GUVs具有控制尺寸，膜组成，膜蛋白掺入和封装3的生成。该方法的基本原理是利用由压电致动器的喷墨变形暂停升产生多个高频流体脉冲IPID双层成GUV。该过程类似于从肥皂膜吹肥皂泡。通过改变喷出的溶液中，涵盖溶液的组成和/或各组分的组合物包括在双层中，研究人员可以应用这种技术来创建定制的囊泡。本文介绍的程序通过microjetting生成简单的小泡从液滴界面双层。

我们已经组建了一个微流体喷射建立在传统的倒置荧光显微镜用自定义的阶段，从加工零件和人工微米组装（ 图1）。喷墨表征提供了洞察囊泡生成过程。不同的喷墨喷嘴和脂质双分子层之间的距离会影响所施加的力，以使膜的变形。靠近双层聚焦的喷流，并防止所述膜从分散能量相差囊泡产生点。涡流行程随施加到压电致动器，与我们的预期（ 图2）相一致的电压。囊?...

Watch this Scientific Journal Video about Lipid Bilayer Vesicle Generation Using Microfluidic Jetting at JoVE.com

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

对液滴界面脂双层微流体喷射提供了一种可靠的方法来产生囊泡膜多不对称，跨膜蛋白的结合，以及材料的封装控制。该技术可用于研究多种生物系统，其中隔离的生物分子是理想的。

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 ...

lipid-bilayer-vesicle-generation-using-microfluidic-jetting

Research

JoVE Journal

Bioengineering

14.8K 次观看.  University of Michigan. 对液滴界面脂双层微流体喷射提供了一种可靠的方法来产生囊泡膜多不对称，跨膜蛋白的结合，以及材料的封装控制。该技术可用于研究多种生物系统，其中隔离的生物分子是理想的。 

视频: Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

恒压控制的挤压方法制备纳米脂质体

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

科研

生物工程

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

生物膜的微阵列用刮墨刀型组合方式形成

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

局部钙离子梯度对大泡囊膜重塑的响应

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

化学

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

单分子荧光显微镜在平面支持双层膜

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

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 ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

聚合物拥挤脂质膜上的单分子扩散和组装

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

生物化学

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip:蛋白质 - 磷酸肌醇相互作用的无标记研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

细胞模拟支持和悬浮脂质双分子层模型的组装用于分子相互作用研究

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.