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）的脂质（重量:ⅴ）在2以下的混合物:8 N -癸烷和十六烷（ν: v）中。隔夜搅拌用旋转器。 </li></ol></li><li>的用于膜形成的脂质溶液的制备: <ol><li>配制制膜的脂质溶液，溶解0.1％DPhPC（1,2-diphytanoyl- SN -glycero -3-磷脂酰胆碱）的脂质（重量:v）的以2:1的混合物:8平方ualene和十六烷（体积:体积）。隔夜搅拌用旋转器。 </li></ol></li></ol> 2.将PDMS薄膜的形成<ol><li>在一个混合杯1（重量/重量）的比例，以形成与PDMS的预聚物:混合的PDMS和固化剂在一个9。 5克PDMS的预聚物添加到培养皿，以形成的PDMS薄膜（厚度200 - 250微米）。使用以800rpm旋涂10秒，以形成一种薄膜传播的PDMS预聚合物。 </li><li>放置培养皿放入真空干燥器在100毫托的压力下2小时以去除气泡。以在70℃聚合的预聚物薄膜，烘烤在烘箱中5小时。 </li><li>为了使一个正方形的PDMS薄膜，切聚合的PDMS薄膜成2×2 平方厘米的正方形。使用500微米的微冲，使一个孔中的PDMS薄膜的中心。预涂料孔与2混合3％DPhPC脂质溶液:8 正-癸烷和十六烷。 </li></ol> 3.商会制作和奥丝mbly <ol><li>来制造BLM室中，使用3D绘图软件用4厘米×1.5厘米×1cm到为1.5厘米×1.3厘米×0.8厘米17内孔的尺寸外尺寸的腔室的设计中有两个对称的块。 </li><li>工艺采用PTFE块以数控机床室，并按照制造商的说明。 </li></ol> 4.商会大会<ol><li>为了组装腔室中，放置两种PTFE块之间的彩涂-PDMS薄膜使得在PDMS薄膜的孔与在腔室中的孔对准。 </li><li>密封采用油脂的盖玻璃（促进光学观察）的腔室的外边缘。固定用螺母和螺栓装配腔室。 注意:确保该室是良好的密封，从而不存在液体泄漏。 </li></ol> 5.前体膜的形成与加急自组装形成（MPES） <ol><li>用吸管，0.5存8 N -癸烷::0.1％DPhPC脂质在2混合微升十六烷到的PDMS薄膜与室组装的孔。 </li><li>在使用之前，该腔室储存在冷冻或低于10℃的冰箱中。 </li></ol> 6.膜形成和验证<ol><li>以形成具有MPES一个BLM，从冰箱退出腔室和暂停将2ml缓冲溶液在腔室中的每一侧。抛开该室&lt;10分钟，直到冻膜前体解冻。 </li><li>腔室放置到一个显微以相对于所述光源和显微镜精确控制的高度。照亮腔室的一侧使用卤素光纤照明器照亮的PDMS薄膜为BLM形成工艺的光学观察的孔径的光源。 </li><li>在另一边，垂直放置的数字显微镜相对于所述光源观察BLM形成（由20放大0X）。 </li><li>以确认BLM的形成，观察其中颜色变得比环形亮的孔的中心。 </li></ol> 7.电气录音<ol><li>电气测量，准备用次氯酸钠一个208微米厚的银线和漂白剂&gt; 1分钟银/氯电极。银/氯电极置于所述室足够深的每一侧被浸入缓冲溶液。 </li><li>电极连接到微电极的放大器。使用电生理学软件，应用±10毫伏三角波形穿过膜以获得一个方波。设置通过点击V_clamp（毫伏）指示的箭头施加电压。 </li><li>通过点击记录按钮（红点图标）记录膜的电性能。直到形成均匀的方波观察记录继续。通过单击黑色方形图标退出录音。 </li></ol> 8.离子通道掺入不E:短杆菌肽A（GA）自发成立后，发生形成BLM，因为GA直接添加到脂质的解决方案。 <ol><li>观察GA信道活动，以5千赫兹的采样速率穿过膜应用100毫伏测量保持该膜的电位。设置通过点击V_clamp（毫伏）指示的箭头施加电压。 </li><li>通过点击录音键（红点图标）记录纳入GA的电气性能。直到电流跳观察进行录音。通过单击黑色方形图标退出录音。 </li><li>电数据采集后，用低通贝塞尔滤波器在100赫兹使用电生理学软件过滤数据。 </li><li>观察当前的跳跃在过滤控股潜在的数据（每个电流跳，0.15〜NS，代表GA离子通道的二聚化）来验证GA结合。 </li></ol>

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The authors have nothing to disclose.

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

automated-lipid-bilayer-membrane-formation-using-polydimethylsiloxane

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

18.1K 次观看.  Inha University. 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。 

视频: Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

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 ...

科研

生物工程

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

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

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 emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

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

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

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 ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

在水/空气界面上掺杂与疏水有机分子混合脂膜的自组装

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

化学

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

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

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays&#160;and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

纳米巴支持的脂质双层系统，用于体外研究膜曲率传感蛋白

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

神经科学

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

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the 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 ...

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 ...