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 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 ...

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

Research

JoVE Journal

Bioengineering

18.0K 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

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

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 can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

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

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

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

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

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

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 ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

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

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 ...

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

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

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

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

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

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 to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

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 ...