作者承认瑞士国家科学基金会资助 SMhardBi (项目编号 200021_150088/1)。在瑞士 Villigen 谢瑞 Instute 的瑞士散裂中子源 SINQ 进行了无 san 实验。作者热烈感谢 Dr. Kohlbrecher 的指导和无实验。在高磁场下的双折射测量装置是从荷兰奈梅亨型磁性实验室 HFML 的现有设置中得到启发的。我们感谢布鲁诺菲斯特的帮助, 开发的电子双折射设置, 1月 Corsano 和丹尼尔 Kiechl 为构建框架允许精细和轻便的激光对准, 和 Dr. 伯纳德科勒为持续的技术支持。

1. DMPC/甲醚-DTPA/tm3 + (摩尔比 4:1: 1) 和 DMPC/-OH/甲醚-DTPA/tm3 + (摩尔比 16:4: 5:5) 多组件的制作程序
<ol>
	<li>初步准备<ol>
		<li>用乙醇稳定的氯仿 (#62; 99% 氯仿) 冲洗所有的玻璃器皿, 用压缩空气烘干。</li>
		<li>在乙醇稳定氯仿 (#62 中生产2种不同的10毫克/毫升的 DMPC 和甲醚-DTPA 的库存溶液; 99% 氯仿), 在乙醇稳定的氯仿 (和 #62; 99% 氯仿) 和10毫米的 TmCl 的库存溶液中, 有一个10毫米的电液.3甲醇.</li>
		<li>在100毫升的超纯 H2O 中, 在 pH 值为7.4 的磷酸二氢钠和0.599 克无水磷酸氢磷酸钠中混合0.121 克, 制备50毫米磷酸盐缓冲液。</li>
	</ol>
	</li>
	<li>干脂膜的制备<ol>
		<li>称量所需数量的分子 (DMPC, 甲醚-DTPA, 并可选的, 哦) 和在3 +库存解决方案在单独的3毫升玻璃杯与2.5 毫升玻璃注射器。<ol>
			<li>对于3毫升的 DMPC/DMPE-DTPA/Tm3+ (摩尔比 4:1: 1, 总脂浓度为 15 mM), 重3.6435 克的 DMPC 股票溶液, 1.4731 克的甲醚 DTPA 库存溶液和0.7126 克 TmCl3股票解决方案。</li>
			<li>对于3毫升的 DMPC/甲醚-DTPA/Tm3 + (摩尔比率 16:4: 5:5、总脂浓度为 15 mM), 重2.9148 克的 DMPC 库存溶液, 1.4731 克甲醚-DTPA 库存溶液, 1.0749 克-OH 股票溶液和0.7126 克TmCl3库存解决方案。 注意: 氯仿和甲醇在室温下具有毒性和挥发性。在通风罩下工作, 并及时进行质量测量。</li>
		</ol>
		</li>
		<li>把杯的内容转移到一个25毫升的圆底烧瓶。将每个杯状水冲洗成圆底烧瓶, 大约2.5 毫升的相应溶剂。</li>
		<li>在40° c 的旋转式蒸发器中, 将溶剂在真空下除去。将初始压力设置为 30 000 Pa, 直到大部分溶剂被除去。将压力降至 100 Pa, 并在旋转条件下将试样干燥至少2小时. 在烧瓶的玻璃墙上获得均匀的干脂膜。</li>
		<li>在稳定的氩气流下将干性脂质膜放置1分钟, 以避免空气中的脂质氧化, 并将样品储存在冷冻前。</li>
	</ol>
	</li>
	<li>干脂膜的水化<ol>
		<li>将3毫升磷酸缓冲液添加到圆底烧瓶, 达到总脂浓度为15毫米。</li>
		<li>进行冻融 (FT) 循环, 将烧瓶在液氮中旋转, 直到彻底冷冻 (液氮停止沸腾), 然后将样品放在水浴中5分钟, 然后再加热到60° c, 不断地旋转烧瓶以帮助熔化过程。应用三十年代的涡流之前, 每个冻结周期, 当样品是液体, 以帮助水合的脂质膜。 注: 第二次冻融循环后, 在烧瓶壁上不应可见脂质膜。</li>
		<li>重复1.3.2 总共五次。在样品热时, 用瓶盖盖上瓶子以避免不必要的磷酸盐缓冲液的蒸发。在冻结样本时, 可以暂停该协议。</li>
		<li>进行两次加热和冷却 (H 和 #38; C) 循环以稳定从最后一个冻结步骤中出来的样品, 或者保持冷冻长达两个月。将样品加热到40或60° c 用于 DMPC/甲醚-DTPA/Tm3 + (摩尔比 4:1: 1) 或 DMPC/甲醚-DTPA/Tm3 + (摩尔比 16:4: 5:5), 在冷却到5° c/分钟之前分别为1°/分. 保持样品5分钟的最大和最小值循环的温度。</li>
		<li>现在, 要么确定在外部磁场中样品的双折射信号 (步骤 2), 要么进一步挤出样品来定制 bicelle 尺寸和磁性 alignability (步骤 1.4)。 注: DMPC/甲醚-DTPA/Tm3 + (摩尔比 4:1: 1) 样品主要由 bicelles 和 70 nm 的平均水动力直径 DH组成, 由在5° c 的动态光散射 (dl) 测量得到的数分布所揭示。这些示例还包含更大的多程序集, 其平均 DH为 500 nm, 显示为强度分布。DMPC/甲醚-DTPA/Tm3 + (摩尔比 16:4: 5:5) 样品是高度多的大小与典型的强度分布, 揭示了平均 DH的 700 nm, 而数字分布显示的人口占主导地位较小bicelles 在 200 nm 的大小范围内。Isabettini et al.报告了这些样品的更详细的尺寸分布和低温透射电镜图像。23</li>
	</ol>
	</li>
	<li>多组件的挤出。<ol>
		<li>组装挤出机, 如图 3所示。使用手套和镊子保护硅胶管处理。用几滴缓冲液将滤纸 (5) 浸湿, 使膜过滤器 (6) 的最佳位置。在顶部放置 o-ring (7) 后, 请确保纸张没有褶皱。 注: 在膜过滤器 (6) 上对挤出过程进行了测试, 孔径为800、400、200和 100 nm;请参见图 7。</li>
		<li>设置水浴到40° c 为 DMPC/甲醚-DTPA/Tm3 + (摩尔比 4:1: 1) 样品或60° c 的 DMPC/甲醚-DTPA/Tm3 + (摩尔比 16:4: 5:5) 样品, 以保证形成可囊泡。</li>
		<li>将挤出机与加压氮气瓶连接, 使用高压 PVC 管 (#62; 4 MPa), 配有 serto 适配器, 并通过膜挤压液体材料。通过膜过滤器 (6) 进行挤出, 通常需要1兆帕斯卡的压力, 孔径为 200 nm 以上。1.5-2.5 MPa 是需要的较小的膜过滤器 (6) 与孔径 100 nm。 注: 如果要求挤出样品 (这是堵塞的第一个迹象), 则需要改变膜过滤器, 如果异常高压 (和 #62; 2.5 MPa)。</li>
		<li>打开盖子 (10) 和插入样品使用2毫升玻璃吸管。然后关闭盖子 (10), 打开压力阀 (12), 同时保持样品出口管 (2)。关闭压力阀 (12), 在挤出循环完成后, 放空并继续下一循环。 注意: 不要把样品留在接触太长的热套管容器 (8), 以避免过多的样品损失的蒸发。</li></ol></li></ol>30-60 s 是足够的时间为3毫升样品平衡在挤出机前打开压力阀 (12)。
		<li>对给定的膜孔尺寸进行10挤出循环, 如图 3所示。大多数的 bicelle 系统通过膜的10倍, 孔直径为200和另10倍, 通过膜的孔径为 100 nm, 保证样品的可比性。</li>
		<li>现在, 在外部磁场中确定样品的双折射信号 (步骤 2)。</li>
	
	

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56812/56812fig3.jpg"> 
图 3: 用于 bicelle 和囊泡制剂的实验室挤出机.挤出机从底部组装: (1) 安装, (2) 样品收集空间与2.4 毫米 (内径) 塑料出口管和 o-ring, (3) 和 (4) 大和小稳定网, (5) 滤纸, (6) 膜过滤器, (7) o-ring, (8) 夹套容器, (9) 顶盖与进口和压力连接, (10) 盖, (11) 蝶形螺丝, (12) 压力阀。装配式挤出机的示意图显示在右手边。氮气 (N2) 由压力容器提供, 套管容器 (9) 连接到水浴以进行温度控制。样品经过10挤出循环, 任何给定的膜过滤孔径 (样品路径显示蓝色)。<a href="//ecsource.jove.com/files/ftp_upload/56812/56812fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 双折射测量 DMPC/甲醚-DTPA/tm3 + (摩尔比 4:1: 1) 和 DMPC/-OH/甲醚-DTPA/tm3 + (摩尔比 16:4: 5:5) 多组件。
<ol>
	<li>生成并连接图 4中所示的双折射设置, 并为各自的电子元素提供电源。在这个阶段不要将 PEM、样品和第二偏振器放在激光路径中。避免通过覆盖反射面 (例如铝镜架) 和黑色纸张来检测后向散射激光。</li>
	<li>调整镜像以使探测器的激光强度最大化, 这由从<img alt="Equation 7" src="/files/ftp_upload/56812/56812eq7.jpg"> 图 4B中的低通入筛选器获得的直流强度表示。 注意: 在调整镜像时要佩戴适当的护眼, 并在第一次操作激光器时与激光安全指导师协商。</li>
	<li>打开第一个交叉的线性偏振器 (与入射激光光束保持垂直) 以最大化. <img alt="Equation 7" src="/files/ftp_upload/56812/56812eq7.jpg"> </img></li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56812/56812fig4.jpg"> 
图 4: 双折射设置和光学信号连接的示意图表示.a)超导电磁铁提供 5.5 T 磁场。二极管激光器在 635 nm 的光被两个交叉的偏振极化。光弹调制器 PEM-90 操作在50赫与振幅一个0 2.405 rad 和放置在两个偏振。样品位于 PEM 和第二偏振器之间的磁铁上。非偏光镜引导光线通过不同的元素, 最后通过一个光电探测器检测到。监测交流信号的第一和第二谐波和, 允许计算在 Ln <img alt="Equation 10" src="/files/ftp_upload/56812/56812eq10.jpg"> <img alt="Equation 11" src="/files/ftp_upload/56812/56812eq11.jpg"> 3 +螯合多组件的磁性 alignability 上提供信息的双折射信号。样品试管连接到外部水浴为温度控制 (蓝色)。用温度探头 (红色) 监测样品的温度。B)通过±12 V 直流电源 braded 电缆 (3), 来自照片探测器的信号被送入第二阶 Sallen 键低通滤波器 (24 v 交流电源), 其截止频率为360赫兹。低通滤波器提取 DC 组件, 并通过 BNC 50 Ω缆线将其传递给 PC 接口 (4). <img alt="Equation 7" src="/files/ftp_upload/56812/56812eq7.jpg">通过 BNC 50 Ω缆线 (1) 和 #38 将照片探测器发出的信号传送到两个锁定放大器 (以提取第一个和第二个谐波和); (2). <img alt="Equation 10" src="/files/ftp_upload/56812/56812eq10.jpg"> <img alt="Equation 11" src="/files/ftp_upload/56812/56812eq11.jpg"> </img>谐波强度检测的相位敏感检测。因此, pem 信号被用作锁定放大器的参考信号 (pem 的 1 f-输出到第一个锁相放大和 2 f 输出到第二, 与 BNC 50 Ω电缆连接)。输出信号通过 BNC 50 Ω缆线传送到 PC 接口单元。模拟采集单元 cFP-AI-110 和 cFP-CB-1 数字化通过 RS 232 缆线传输到计算机的信号进行监视。类型 K 温度探头还连接到 PC 接口单元, 其中模拟采集单元 cFP-CB-3 和 cFP-TC-120 数字化信号, 然后通过 RS 232 缆线将其传送到计算机以进行监测。C) 在 b. 关键要素中提出的示意图设置的图片从1到4对应的数字进行识别。<a href="//ecsource.jove.com/files/ftp_upload/56812/56812fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="4">
	<li>将第二个交叉线偏振器垂直于入射激光光束, 如图 4A所示。通过将第二个偏光片设置为第一个90°角度来最小化. <img alt="Equation 7" src="/files/ftp_upload/56812/56812eq7.jpg"></li>
	<li>将光弹性调制器 (PEM) 置于两个交叉线性偏振之间的0°之间, 垂直于入射激光束, 如图 4A所示。如图 5A所示, 将 PEM 调整为50赫和振幅 A0 到 2.405 rad 的频率。这使得 DC 组件独立于双折射, 并增强了. <img alt="Equation 7" src="/files/ftp_upload/56812/56812eq7.jpg"> </img> 注意: 在测量任何样品之前, PEM 的光轴可以通过几度来调节, 以保持空气中的恒定的. <img alt="Equation 7" src="/files/ftp_upload/56812/56812eq7.jpg"></li>
	<li>在打开激光和电子设备后等待1小时, 以稳定信号。</li></ol>一旦锁定放大器的 auto-phasing 保持不变, 信号就会稳定。
	<li>将样品放在温度控制的石英试管中, 路径长度为10毫米, 并将其连接到最初设置在5° c 的外部水浴。</li>
	<li>在样品中直接放置一个0.5 毫米厚的 K 型热电偶 (温度探头) 来监测样品的温度。通过在激光路径中放置一张白纸 (在试管后) 并寻找由探头引起的阴影, 验证探头是否不干扰激光光源。 注: 水浴温度记录与样品温度之间有2-3 ° c 的差异。</li>
	<li>将试管置于磁体的孔中, 如图 4A所示。激光光通过样品水平传播, 被 non-polarizing 的反射镜所偏转, 并被照片探测器检测到。 注: 激光被定向下来, 通过样品, 并备份相同的路径, 以解释法拉第的影响 (即在下降时, 磁场的极化平面的旋转被取消时, 返回在相反的方向)。</li>
	<li>在室温和 10000 Pa 的试管上采用稳定的压缩空气气流, 以避免在细胞壁上凝结水分, 从而降低信号强度, 增加噪音。这是特别重要的, 当测量在5° c。</li>
	<li>用两个锁定放大器检测交流信号的第一和第二谐波和. <img alt="Equation 10" src="/files/ftp_upload/56812/56812eq10.jpg"> <img alt="Equation 11" src="/files/ftp_upload/56812/56812eq11.jpg">通过按图 5B中所示的按钮 (2) 自动相位锁定放大器, 并按图 5B (1) 所示调整灵敏度。确保放大器上的红条不超过四, 如图 5B (3) 所示, 以避免信号过载。在程序Tesla_Magnet_Const_V092中记下两个锁定放大器的使用灵敏度, 如图 5C (8) 所示。该程序作为补充信息提供。</li>
	<li>通过程序Tesla_Magnet_Const_V092向磁铁提供电流, 从而使磁场上升到 5.5 T, 如图 5C (5) 所示。</li>
	<li>使用公式2获取双折射, 其中的延迟计算方法为<img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> 
	<img alt="Equation 13" src="/files/ftp_upload/56812/56812eq13v2.jpg">(3) 其中, 和是第一种的贝塞尔函数, 其中有和. <img alt="Equation 14" src="/files/ftp_upload/56812/56812eq14.jpg"> <img alt="Equation 15" src="/files/ftp_upload/56812/56812eq15.jpg"> <img alt="Equation 16" src="/files/ftp_upload/56812/56812eq16.jpg"> <img alt="Equation 17" src="/files/ftp_upload/56812/56812eq17.jpg"> </img>11,13,18,33,34在程序Tesla_Magnet_Const_V092中绘制延迟, 如图 5C (4) 所示。 注: 如果两个锁相放大器不以相同的灵敏度运行, 则不应使用程序提供的延迟来计算双折射信号 (参见步骤 2.12)。记录的谐波强度和必须乘以锁定放大器的灵敏度才能得到正确的尺寸. <img alt="Equation 10" src="/files/ftp_upload/56812/56812eq10.jpg"> <img alt="Equation 11" src="/files/ftp_upload/56812/56812eq11.jpg">此外, 在磁体场下测量的双折射信号必须通过减去 0 T 上获得的平均双折射信号进行归一化。</li>
	<li>通过调节连接到图 4中所示的试管的水浴的温度, 在恒定或变化的温度 (1 ° c/分钟) 内监控样品的双折射信号。</li>
	<li>通过在图 5C (8) 中填写实验说明来记录实验数据, 并在 (9) 中提供一个文件名, 并按下 "开始日志" 按钮 (10)。</li>

<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56812/56812fig5.jpg"> 
图 5: 使用的设置和程序截图的插图.A) PEM 设置: 延迟 2.405 rad, 波长 635 nm, 频率50赫兹. 白色圆圈表示要激活的设置 (用户定义的延迟, 即本地操作模式)。B)锁定放大器设置。灵敏度 (1) 必须在每个测量前选择, 如步骤2.11 所要求的那样。在显示器 (3) 上不应有超过四红条, 以避免信号过载。当 (1) 中的红色指示灯打开时, 就会发生过载, 使得测量变得不可能。在每次测量前按自动相位按钮 (2)。C)作为补充信息提供的程序Tesla_Magnet_Const_V092的屏幕截图。该程序允许控制的磁场和记录的所有信号输出作为一个时间的功能。在 (1) 中绘制了磁场强度和试样温度。在 (2) 中绘制了两个锁定放大器测量的交流信号的第一和第二谐波和. <img alt="Equation 10" src="/files/ftp_upload/56812/56812eq10.jpg"> <img alt="Equation 11" src="/files/ftp_upload/56812/56812eq11.jpg">直接电流的强度在 (3) 中绘制. <img alt="Equation 7" src="/files/ftp_upload/56812/56812eq7.jpg">延迟计算如步骤2.13 所述, 并在 (4) 中绘制。磁场强度设置在 (5)。在 (6) 和 (7) 中的输出信号 (和) 中, 直接测量 K 型热电偶记录的温度.<img alt="Equation 23" src="/files/ftp_upload/56812/56812eq23.jpg"> </img> <img alt="Equation 22" src="/files/ftp_upload/56812/56812eq22.jpg"> </img>附加的样本信息可以插入 (8), 如放大器的使用灵敏度, 样品名称等。数据可能被记录并导出到 (9) 中提供的 .txt 文件中。使用 "开始日志" 按钮 (10) 启动和停止数据获取。<a href="//ecsource.jove.com/files/ftp_upload/56812/56812fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<ol>
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作者没有什么可透露的。

详细介绍了双折射测量如何与 san 结合使用, 以评估产生高磁响应的方法在3 +螯合磷脂组件中的 Isabettini et al.23所建议的制造协议也适用于由较长的联邦和 DPPE-DTPA 磷脂组成的组件, 或用于含有其双层内化学工程类固醇衍生物的部件。11,12,17,18,19...

Bicelles 是从许多脂质混合物中获得的圆盘状多组件。1,2,3,4,5广泛应用于膜生物分子的核磁共振波谱结构表征。6,7然而, 最近的努力旨在扩大可能的应用领域。5,8,9最受研究的 bicelle 系统由 12-二-sn-glycero-3-phosphocholine (DMPC) 组成, 构成组件的平面部分, 12-己-snglycero-3-phosphocholine (DHPC)磷脂覆盖边缘。1,2,3组成双层磷脂的分子几何决定了自组装多结构的结构。4,5用甲醚-DTPA 替换 DHPC, 生成高磁响应和可调谐的 bicelle 系统。10,11 DMPC/甲醚-DTPA/Ln3 + (摩尔比 4:1: 1) bicelles 与更多的顺磁性镧系离子 (Ln3 +) 在双层表面上相关联, 从而增强了磁响应。10此外, 用甲醚-DTPA/Ln3 +取代水溶性 DHPC 分子, 可以形成耐稀释的 bicelles。11
平面多组件的磁性 alignability 是由它们的整体磁场能量决定的,
<img alt="Equation 1" src="/files/ftp_upload/56812/56812eq1v2.jpg">(1)
其中 B 是磁场强度, 磁常数, n 的聚合数和分子磁性的易感性各向异性组成双层的脂质. <img alt="Equation 2" src="/files/ftp_upload/56812/56812eq2.jpg"> <img alt="Equation 3" src="/files/ftp_upload/56812/56812eq3.jpg">因此, DMPC/甲醚-DTPA/Ln3 + bicelles 对磁场的响应是根据它们的大小 (集合数 n) 和分子磁性磁化率各向异性Δχ来调整的。后者很容易通过改变螯合物的性质在3 +中实现。12,13,14,15在双层中引入胆固醇 (铁-OH) 或其他甾体衍生物, 可以调节聚合数 n 和Δχ的磁化率。11,16,17,18,19对于给定的脂质组合, 较大的组件含有更多的脂类, 能够对 Emag (更大的聚合数 n) 做出贡献, 从而导致更多的 alignable 物种。DMPC/DHPC bicelles 的大小, 例如, 是常规控制通过优化的组成脂质比或总浓度。20,21,22虽然这在 DMPC/甲醚-DTPA/Ln3 + bicelles 中是可能的, 但在加热时, 它们从 bicelle 到泡的热可逆转换提供了更多的剪裁选项。机械手段, 如挤压通过膜过滤器, 可以形成的囊泡。磁性 alignable bicelles 在冷却到5° c 时再生, 并且他们的维度从泡前体被口授。11在这里, 我们专注于机械制造过程的潜力, DMPC/甲醚-DTPA/tm3 + (摩尔比率 4:1: 1) 或 DMPC/, OH/甲醚-DTPA/tm3 + (摩尔比 16:4: 5:5) 作为参考系统。在使用其他 Ln3 +而不是 Tm3 +时, 该进程工作类似。这些技术提供的各种可能性在图 1中得到了突出显示, 并在其他地方进行了广泛的讨论。23
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56812/56812fig1.jpg"> 
图 1: 可能的制造过程的示意图概述.所研究的磁性 alignable 在3 +螯合多组件是由 DMPC/甲醚-DTPA/Tm3 + (摩尔比 4:1: 1) 或 DMPC/, OH/甲醚-DTPA/Tm3 + (摩尔比 16:4: 5:5) 组成。干脂膜是水合的50毫米磷酸缓冲液 pH 值为 7.4, 总脂质浓度为15毫米。有效的脂质膜水化需要的是冻融循环 (FT) 或加热和冷却循环 (H & #38; C)。H 和 #38; C 周期是必要的, 以再生样品后, 最后冻结解冻步骤, 或再生样品保持长期冻结超过一段时间, 如果他们要使用不进一步挤压。Isabettini et al广泛讨论了这些步骤。23最大的 alignable 多组件实现, 根据脂质组成提供不同的组件体系结构。通过纳米膜过滤器, 挤出 (Ext) 可调节 bicelle 尺寸和磁性 alignability。所呈现的对齐系数f是从2D 小角中子散射 (san) 模式 (DMPC/甲醚/DTPA/Tm3 + (摩尔比率 16:4: 5:5) 中计算的, 通过800、400、200或 100 nm 孔隙进行拉伸。san 度量是量化 bicelle 对齐的补充方法, 此处不会详细介绍。11,16一个f范围从-1 (平行中子散射或垂直对准的 bicelles 与磁场方向) 到0的各向同性散射。<a href="//ecsource.jove.com/files/ftp_upload/56812/56812fig1large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
bicelles 的结构在广泛的表征技术中得到了广泛的研究。13使用核磁共振波谱法或小角度中子散射 (san) 实验对暴露在磁场中的 bicelles 的对准进行了量化。5,10,11,12,13,16,17,18,19,24,25但是, 在 Ln3 +的情况下发生的核磁共振峰值的移位和展宽是对该方法的严重限制。15,26,27,28尽管 san 实验不受此限制的影响, 但在解决方案中对组件的磁致对准的常规定量化方面, 还是可取的替代方法。双折射测量是一个可行的和比较简单的选择。类似核磁共振实验, 双折射测量揭示了有价值的信息, 脂质重和脂质相发生在双层。此外, 多组件中发生的几何变换, 如温度等变化的环境条件进行了监测。11,12,13,16磁致双折射Δn′用于研究各种类型的磷脂系统。13,29,30基于磁场相位调制技术的双折射测量是检测 bicelles 方位的可行方法。12,16,18,29,31,32在高磁场中 bicelles 的双折射率高达 35 T 的可能性也由 m. Liebi et al.显示13
当偏振光进入一个各向异性物质时, 它会在一个普通的和特殊的波中折射。11两个波具有不同的速度, 并在相位上由一个迟滞δ转移。延迟δ的程度被测量并且转换成双折射信号来量化材料中各向异性的程度<img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg">
<img alt="Equation 6" src="/files/ftp_upload/56812/56812eq6.jpg">(2)
其中λ是激光的波长, d 是样品的厚度。磷脂是光学的各向异性, 它们的光轴与它们的长分子轴重合, 与烃尾平行。11,12如果磷脂在溶液中是随机定向的, 则不测量延迟。当磷脂相互平行时, 就会测量迟钝。磁致双折射可根据磁场中分子的取向, 产生正负符号; <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg">请参见图 2。与 x 轴平行的磷脂将导致一个负的, 而那些沿 z-axis 对齐结果为正值的. <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> </img> <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> </img>当与 y-axis 平行时, 当光学轴与光传播的方向重合时, 不观察到双折射。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56812/56812fig2.jpg"> 
图 2: 磷脂的对准和磁致双折射的对应符号 <img alt="Equation 12" src="/files/ftp_upload/56812/56812eq12.jpg"> </img> .测量的的符号取决于在磁场中磷脂的方向. <img alt="Equation 12" src="/files/ftp_upload/56812/56812eq12.jpg">虚线表示分子的光轴。光在45°上极化, 并在 y 方向传播。磁场 B 在 z 方向。这一数字已从 m Liebi 中修改。11 <a href="//ecsource.jove.com/files/ftp_upload/56812/56812fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在 bicelles 的各向同性胶体悬浮的情况下, 磷脂在双层中的排列所诱导的取向将会丢失, 使迟滞δ零化。bicelles 还必须对准, 以便在它们的双层中定位光学活性磷脂, 导致偏振光δ。因此, 双折射是一个敏感的工具, 量化的磁性 alignability 的多组件。垂直于磁场的 Bicelles 将产生一个正的, 而那些对齐的平行将产生一个负的. <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> </img> <img alt="Equation 5" src="/files/ftp_upload/56812/56812eq5.jpg"> </img>该符号取决于设置的对齐方式, 并且可以使用引用示例进行检查。

<table><tbody><tr><td>1,2-dimyristoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DMPC)</td><td>Avanti Polar Lipids</td><td>850345P</td><td>&gt;99%</td></tr><tr><td>1,2-dimyristoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate acid hexammonium salt (DMPE-DTPA)</td><td>Avanti Polar Lipids</td><td>790535P</td><td>&gt;99%</td></tr><tr><td>Thulium(III) chloride</td><td>Sigma-Aldrich</td><td>439649</td><td>anhydrous, powder, 99.9% trace metals basis</td></tr><tr><td>Dysprosium(III) chloride</td><td>Sigma-Aldrich</td><td>325546</td><td>anhydrous, powder, 99.9% trace metals basis</td></tr><tr><td>Ytterbium(III) chloride</td><td>Sigma-Aldrich</td><td>439614</td><td>anhydrous, powder, 99.9% trace metals basis</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>319988</td><td>contains ethanol as stabilizer, ACS reagent, &amp;ge;99.8%</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td>&amp;ge;99.9%</td></tr><tr><td>Cholesterol</td><td>Amresco</td><td>433</td><td>Ultra pure grade</td></tr><tr><td>D2O</td><td>ARMAR chemicals</td><td>1410</td><td>99.8 atom % D</td></tr><tr><td>Ultrapure water</td><td>Millipore</td><td></td><td>Synergy pak2 (SYPK0SIX2), Millipack GP (MPGP02001)</td></tr><tr><td>electronic pH meter</td><td>Metrohm</td><td>17440010</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 100nm membrane filter</td><td>VWR</td><td>515-2028</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 200nm membrane filter</td><td>VWR</td><td>515-2029</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 400nm membrane filter</td><td>VWR</td><td>515-2030</td><td></td></tr><tr><td>Whatmann Nuclepore 25 mm 800nm membrane filter</td><td>VWR</td><td>515-2032</td><td></td></tr><tr><td>Whatmann Filter paper</td><td>VWR</td><td>230600</td><td></td></tr><tr><td>25 ml round bottom flask</td><td>VWR</td><td>201-1352</td><td>14/23 NS</td></tr><tr><td>3 ml glass snap-cup</td><td>VWR</td><td>548-0554</td><td>ND18, 18x30mm</td></tr><tr><td>2.5 ml glass syringe</td><td>Hamilton</td><td></td><td></td></tr><tr><td>Sodium dihydrogen phosphate dihydrate</td><td>Merk</td><td>1.06342</td><td>Salt used to make phosphate buffer</td></tr><tr><td>di-Sodium hydrogen phosphate</td><td>Merk</td><td>1.06586</td><td>Salt used to make phosphate buffer</td></tr><tr><td>Liquid Nitrogen</td><td>Carbagas</td><td>-</td><td></td></tr><tr><td>Pressurized Nitrogen gas</td><td>Carbagas</td><td>-</td><td>200 bar bottle</td></tr><tr><td>Lipid Extruder 10 ml</td><td>Lipex</td><td>-</td><td>Fully equipped with thermobarrel</td></tr><tr><td>High-pressure PVC tube</td><td>GR NETUM</td><td>-</td><td>must resist more than 4 MPa</td></tr><tr><td>Serto adaptors</td><td>Sertot</td><td>-</td><td></td></tr><tr><td>Nitrile gloves</td><td>VWR</td><td>-</td><td></td></tr><tr><td>2 ml glass pipettes</td><td>VWR</td><td>612-1702</td><td>230 mm long</td></tr><tr><td>Diode Laser</td><td>Newport</td><td>LPM635-25C</td><td></td></tr><tr><td>DSP Dual Phase Lock-in Amplifier</td><td>SRS</td><td>SR830</td><td></td></tr><tr><td>Photodiode Detector</td><td>Silonex Inc.</td><td>SLSD-71N5</td><td>5mm2, Silicon, photo-conductive</td></tr><tr><td>5.5 T Cryogenic Magnetic</td><td>Cryogenic/Oerlikon AG</td><td>-</td><td>12 bar He-cooled. RW4000/6000 compressor, RGD 5/100 TA cryo-head</td></tr><tr><td>Second order low pass filter</td><td>home-built</td><td>-</td><td>Linear power supply 24V DC, second order, Sallen Key, cut-off frequency 360 Hz, +/- 12V, max 10 mA</td></tr><tr><td>Photoelastic modulator</td><td>Hinds instruments</td><td>PEM-90</td><td></td></tr><tr><td>Glan-Thompson Calcite Polarizer</td><td>Newport</td><td>10GT04</td><td>25.4mm diameter</td></tr><tr><td>Quartz sample cuvette</td><td>Hellma</td><td>165-10-40</td><td>temperature controlled cell, 0.8 ml, 10mm path length</td></tr><tr><td>Temperature probe</td><td>Thermocontrol</td><td>-</td><td>Type K, 0.5mm diameter, Thermocoax</td></tr><tr><td>Non-polarizing mirrors</td><td>Newport</td><td>50326-1002</td><td>25.4mm</td></tr><tr><td>RS 232 cables</td><td>National Instruments</td><td>189284-02</td><td>For Connecting to the RS-232 Port on the front of Compact FieldPoint Controllers</td></tr><tr><td>BNC 50 &amp;Omega; cable and connectors</td><td>National Instruments</td><td>763389-01</td><td></td></tr><tr><td>cFP-AI-110</td><td>National Instruments</td><td>777318-110</td><td>8-Channel Analog Voltage and Current Input Module for Compact FieldPoint</td></tr><tr><td>cFP-CB-1</td><td>National Instruments</td><td>778618-01</td><td>Integrated Connector Block for Wiring to Compact FieldPoint I/O</td></tr><tr><td>cFP-CB-3</td><td>National Instruments</td><td>778618-03</td><td>Integrated Isothermal Connector Block for Wiring Thermocouples to the cFP-TC-120 Module</td></tr><tr><td>cFP-TC-120</td><td>National Instruments</td><td>777318-120</td><td>8-Channel Thermocouple Input Module for Compact FieldPoint</td></tr><tr><td>cFP-1804</td><td>National Instruments</td><td>779490-01</td><td>Ethernet/Serial Interface for NI Compact FieldPoint</td></tr><tr><td>LabView 2010</td><td>National Instruments</td><td>-</td><td></td></tr><tr><td>Industrial power supply</td><td>Traco Power</td><td>TCL 060-124</td><td>100-240V AC</td></tr><tr><td>Waterbath</td><td>Julabo</td><td>FP40-HE</td><td>refrigerated/Heating Circulator</td></tr></tbody></table>

fabrication procedures and birefringence measurements for designing magnetically responsive lanthanide ion chelating phospholipid assemblies

Bicelles 是可调谐的圆盘状多组件, 由大量的脂质混合物组成。应用范围从膜蛋白结构研究的核磁共振 (NMR), 以纳米的发展, 包括形成光学活性和磁性可转换凝胶。这些技术需要对装配尺寸、磁响应和热阻进行高度控制。混合物 12-二-sn-glycero-3-phosphocholine (DMPC) 及其镧系离子 (Ln3 +) 螯合磷脂共轭, 12-二-snglycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (甲醚-DTPA), 组装成高磁响应的组件, 如 DMPC/甲醚-DTPA/Ln3 + (摩尔比率 4:1: 1) bicelles。在双层中引入胆固醇 (-OH) 和类固醇衍生物会导致另一组提供独特物理化学性质的组件。对于某一特定的脂质成分, 磁性 alignability 与 bicelle 大小成正比。在3 +中的络合结果在大小和对准方向方面都产生了前所未有的磁响应。热可逆崩溃的圆盘状结构成泡后, 加热允许裁剪装配的尺寸, 通过挤出膜过滤器的定义孔径大小。磁性 alignable bicelles 通过冷却到5° c 再生, 导致装配尺寸由囊泡前体定义。在此, 解释了这个制造过程, 并通过在 5.5 T 磁场下的双折射测量来量化组件的磁性 alignability。双折射信号, 源自磷脂双层, 进一步使监测的多变化发生在双层。这种简单的技术是互补的核磁共振实验, 通常用于表征 bicelles。

non-extruded DMPC/甲醚-DTPA/Tm3 +的双折射信号 (摩尔比 4:1: 1) 在一个 5.5 T 磁场的加热和冷却循环期间, 从5到40° c 和背部以1° c/分钟 (图 6) 的速度监测样品。双折射结果在5° c 时确认了高磁对齐, 其值为 1.5 x 10-5, 为所报告的挤出系统的两倍强。6,7,23 DMPC 在24° c 以上的 T<s...

Watch this Scientific Journal Video about Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies at JoVE.com

Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

介绍了高磁响应稀土离子螯合多组件的制备方法。磁响应由装配尺寸决定, 它是通过纳米膜的挤压而定制的。采用双折射测量、核磁共振和小角度中子散射技术对组件的磁 alignability 和温度诱发的结构变化进行监测。

设计磁响应稀土离子螯合磷脂组件的制备方法及双折射测量

Bicelles are tunable disk-like polymolecular assemblies formed from a large variety of lipid mixtures. Applications range from membrane protein structural ...

fabrication-procedures-birefringence-measurements-for-designing

Universidad de Cartagena UDEC

Research

JoVE Journal

Engineering

7.1K 次观看.  ETH Zurich. 介绍了高磁响应稀土离子螯合多组件的制备方法。磁响应由装配尺寸决定, 它是通过纳米膜的挤压而定制的。采用双折射测量、核磁共振和小角度中子散射技术对组件的磁 alignability 和温度诱发的结构变化进行监测。

视频: Fabrication Procedures and Birefringence Measurements for Designing Magnetically Responsive Lanthanide Ion Chelating Phospholipid Assemblies

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

定点自旋标记和五聚体配体门控离子通道的EPR光谱研究

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

科研

化学

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

类囊脂质体中硅藻Cyclotella Meneghiniana中的光捕获天线的分离与融合

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

生物化学

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

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 ...

生物工程

Measuring Magnetically-Tuned Ferroelectric Polarization in Liquid Crystals

Materials showing coupling phenomena between magnetism and (ferro)electricity, i.e., magnetoelectric effects, have attracted a great deal of attention due to their potential applications for future device technologies such as sensors and storage. However, conventional approaches, which usually utilize materials containing magnetic metal ions (or radicals), have a major problem: only a few materials have been found to show the coupling phenomena at room temperature. Recently, we proposed a new approach to achieve room-temperature magnetoelectrics. In contrast to conventional approaches, our alternative proposal focuses on a completely different material, a &#34;liquid crystal&#34;, free from magnetic metal ions. In such liquid crystals, a magnetic field can be utilized to control the orientational state of constituent molecules and the corresponding electric polarization through magnetic anisotropy of the molecules; it is an unprecedented mechanism of the magnetoelectric effect. In this context, this paper provides a protocol to measure ferroelectric properties induced by a magnetic field, that is, the direct magnetoelectric effect, in a liquid crystal. With the method detailed here, we successfully detected magnetically-tuned electric polarization in the chiral smectic C phase of a liquid crystal at room temperature. Taken together with the flexibility of constituent molecules, which directly affects the magnetoelectric responses, the introduced method will serve to allow liquid crystal cells to acquire more functions as room-temperature magnetoelectrics and associated optical materials.

In this report, we present a protocol to examine direct magnetoelectric effects, i.e., induction of ferroelectric polarization by applying magnetic fields, in liquid crystals. This protocol provides a unique approach, supported by the softness of liquid crystals, to achieve room-temperature magnetoelectrics.

液晶中磁调谐铁电极化的测量

Materials showing coupling phenomena between magnetism and (ferro)electricity, i.e., magnetoelectric effects, have attracted a great deal of attention due ...

工程学

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 ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

水样中偶极辅助固相萃取Microchip的痕量金属分析的制作

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

基于荧光的测量磷酸酯/磷酸二醇 4-磷酸盐膜之间的交换

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

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 ...

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 ...