部分支持DS和CEC的授权AI053531（NIAID，NIH）; SS和PSC由授权N00014-14-1-0792（ONR）支持。

1.清洁玻璃盖板<ol><li>稀释7倍的清洁溶液（参见材料表 ）用100毫米深硼硅酸盐玻璃皿的去离子水将其稀释7倍，并将其在平板热板上加热至95℃，持续20分钟，或直到混浊溶液变澄清。 注意:解决方案将会很热，请注意避免身体受伤。 7x清洁溶液是[氧化 - （氧化（膦酰氧基）磷酰基]氧磷酰基]磷酸六钠，2-（2-丁氧基乙氧基）乙醇，1,4-双（2-乙基己氧基）-1,4-二氧代丁烷钠-2-磺酸盐和无害添加剂。 </li><li>使用镊子，在铝染色架上沿交替方向排列盖玻片（40毫米长x 22毫米宽×0.16-0.19毫米高）。在每个盖玻片之间保持空位，以防止它们彼此接触。 </li><li>将染色架完全淹没在清洁溶液中。保留盖玻片解决方案1小时。 注意:当水蒸发时，继续加入新鲜的去离子水以保持溶液浓度不变。 </li><li>将染色架从清洁溶液中取出，并用大量的去离子水清洗盖玻片以除去洗涤剂。 </li><li>用氮气干燥盖玻片（每个染色架约5分钟），然后将盖玻片放置在窑炉中550℃下6小时退火。 注意:这是平滑玻璃表面粗糙特征的关键步骤。 </li><li>将盖玻片放在塑料容器中，使其远离灰尘。 </li></ol> 2.制作微图案PDMS块<ol><li>在大塑料称重舟皿中以10:1（w / w）的比例混合聚二甲基硅氧烷（PDMS）预聚物和固化剂，并在真空中真空脱气1小时，真空强度为500托或更低。 注意:PDMS是一种透明和惰性的硅氧烷聚合物。获得所需的PDMS块t（0.5厘米），将55克预聚物与5.5克固化剂混合。 </li><li>将含有相同SU-8微图案的多个重复的硅母版置于大（10厘米基径）的塑料称重船中，并倒入脱气的PDMS中。然后，在60℃的干燥炉中固化过夜。 注意:微图案尺寸足够大，可以用肉眼看到。每个图案包含八个微通道（100μm宽×40μm高×1cm长），间隔40μm（ 图1A ， 1B ）。在纳米加工设备27中制造由硬烘烤的SU-8光致抗蚀剂组成的硅晶片模具。对于主制备的其它方法包括氢氟酸（HF）蚀刻，高温水腐蚀，xurography和3D印刷28，29，30，31。 COMMERC也可以使用硅制备的主要来源。微流体装置的图案设计有起草软件（见材料表）。包含图案设计的原始文件作为补充文件"Pattern Design.dwg"提供，可以直接提供给制造商。 </li><li>轻轻地用手剥离硅胶主板上的PDMS。使用外科手术刀和尺子标记矩形中每个微图案的边界。然后，将PDMS切成块。 </li><li> 如图3B所示 ，每个微通道的两端用活检穿孔（1.0mm孔直径）打入16孔（每块8个入口和8个出口）。 </li><li>在每个PDMS块上涂抹胶带，以保护微图案免受损坏和灰尘。将它们存放在塑料容器中。 </li></ol> 3.准备小单层囊泡（SUVs） 注意:阴性对照双层组成是99.5mol％1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）和0.5mol％ 邻磺酰罗丹明B-1-棕榈酰-2-油酰-sn-甘油-3-磷酸乙醇胺（ o SRB-教皇）。测试双层组成为92.0mol％POPC，0.5mol％ o SRB-POPE和7.5mol％的L-α-磷脂酰肌醇-4-磷酸（PI4P）或L-α-磷脂酰肌醇-4,5-二磷酸酯（PI（ 4,5）P 2 ）。以下是制备92.0mol％POPC，0.5mol％ o SRB-POPE和7.5mol％含PI（4,5）P 2的SUV的方法。 øSRB-POPE在本研究中使用的合成先前被描述20。 <ol><li>计算需要混合的POPC，PI（4,5）P 2和o SRB-POPE的体积，占以下总量:2.22mg POPC，0.26mg PI（4,5）P 2 ，和2.1×10 -5毫克邻 SRB-POPE的。 </li><li>吸取计算的POPC体积，PI（4,5）P 2 ，&lt;将SRB-POPE注入单个20mL玻璃闪烁瓶中。 注意:POPC和o SRB-POPE储备溶液含有氯仿溶液，而PI（4,5）P 2 （和其他磷酸肌醇）储备液中加入氯仿:甲醇:水（20:9:1）溶剂混合物。为了准确，使用前用氯仿将吸液管吸干。为安全起见，此步骤应在化学通风柜内进行。 </li><li>将混合物在化学通风柜内的氮气流中干燥10分钟，或直到溶剂蒸发，并在小瓶的底部形成薄的脂质膜。 </li><li>在10 mTorr的真空强度下真空干燥混合物≥3h，以除去任何残留的有机溶剂。 注意:3小时干燥时间足以满足本协议中描述的SUV制剂规模，这足以满足50次实验。如果需要更大规模的SUV制备，则可以进行隔夜干燥。 </li><li>再加水用20mL HEPES，100mM NaCl，pH7.0的5mL运行缓冲液将其置于35kHz的操作频率的超声波浴中30分钟。 注意:混合步骤3.1中指定的脂质的量并在步骤3.5中加入5mL的运行缓冲液，得到0.5mg / mL的囊泡悬浮液。在这个阶段，囊泡悬浮液是单层和多层囊泡的混合物（ 图2B ）。该解决方案将显示为粉色/紫色，相对浑浊。 </li><li>用液氮和水浴在40℃下冻融囊泡悬浮液以获得单层囊泡。重复冻融10次。 注:（可选）以确保不存在在悬浮液中的非单层囊泡的，离心步骤可以应用32,33。 </li><li>使用脂质挤出机将小泡悬浮液通过0.10μm轨道蚀刻的聚碳酸酯膜挤出（参见表格）丰富了SUV。重复挤压10次。 注意:可以使用15-mL锥形管来收集挤出的囊泡。该溶液在此阶段应无浊度。可以通过动态光散射（DLS）确认SUV直径（ 图2C ）。 SUV最适合于形成SLB 34 。 </li><li>用铝箔盖住锥形管，并保存在4°C直到准备使用。 </li></ol> 4.组装微流控装置<ol><li>通过使用水洗瓶将去离子水喷射穿过孔，测试PDMS块的入口和出口堵塞。然后用氮气干燥PDMS块。 注意:此步骤还将清除可能已被捕获在通道内和/或通道之间的任何灰尘颗粒。如有必要，用氮气除尘预清洁的盖玻片，以除去任何灰尘颗粒。 </li><li>放置PDMS块并预先清洁氧气等离子体系（见表格）样品室内的盖玻片（见第1节），用氧等离子体暴露45秒，功率设定为75瓦，氧气流速为10厘米3 /分，真空强度为200毫托。 </li><li>在氧等离子体处理后立即将PDMS块的图案化表面立即与盖玻片接触。轻轻按压以清除接触部位的任何气泡。 </li><li>将设备放置在100℃的热平板上3分钟以增强粘合。 </li><li>使用带有100％乙醇的湿式无绒毛擦拭（ 例如 kimwipe）从设备的顶部（PDMS）和底部（玻璃）中除去任何灰尘颗粒。然后，将设备贴在玻璃显微镜载玻片的顶部。 注意:不要使用过量的乙醇，并避免乙醇进入入口和出口通道。建议在设备仍然热的情况下执行此步骤，以确保乙醇立即蒸发。玻璃微型振荡玻片可以储存在100％乙醇溶液中，以除去灰尘和其他污染物。 </li></ol> 5.形成支持的脂质双层（SLB） <ol><li>将100μL来自步骤3.8的PI（4,5）P 2的SUV转移到0.65mL的微量离心管中。通过加入6.4μL0.2N盐酸将溶液的pH调节至〜3.2。 注意:用配有微量pH探针的pH计确认溶液的pH值（参见表格）。 </li><li>通过入口将10μLpH调整后的SUV溶液吸入每个通道，并通过移液管施加压力，直到溶液到达出口。将吸头从吸液管中取出并将其连接到设备上。 </li><li>对每个通道重复上述步骤，然后在室温下孵育10分钟。 注意:在器件组装后，应立即执行将囊泡注入微通道。 </li><li>入口和出口管的切割套件ea长60厘米（入口管）和8厘米（出口管）长。 注意:对角切割管道并创建锋利的边缘，可以更轻松地将管道插入入口和出口。出口管应具有弧形。入口管的长度可以根据显微镜设置而变化。管的内径为0.05厘米。 </li><li>使用镊子将出口管道连接到设备，然后将设备粘贴到显微镜平台上。 </li><li>将入口管的一端浸没在25 mL运行缓冲液中，包含在锥形管中，并将其胶带，以确保管道固定。 </li><li>将锥形管放置在比设备更高的地面（约20厘米）上，以便通过重力流将溶液推入微通道;可以使用实验室插孔来实现这一点。 </li><li>对于每个入口管，使用注射器从管道的自由端抽出1 mL运行缓冲液。从进样口取下移液器吸头并插入入口管的自由端进入设备。 注意:将一滴运行的缓冲液引入入口以减少在此步骤期间气泡被引入通道的可能性。入口管连接到设备后，使用无毛巾擦去多余的缓冲液。 </li><li>重复上述步骤将所有入口管件连接到设备。 注意:通过通道的流动缓冲液有助于去除过量的囊泡，并将双层平衡到实验条件。 </li><li>打开显微镜控制软件（见表格材料）。在左面板上，点击"显微镜"选项卡，选择"10X"目标。 </li><li>点击工具栏上的"Live"，然后点击"Alexa 568"图标图标。使用精细和校准调节旋钮，专注于微通道。 </li><li>扫描设备以检查SLB和通道的质量（ 图8 ）。然后，点击工具栏上的"FL快门关闭"图像图标。 </li><li>点击"采集"选项卡，在"基本调整"下选择"曝光时间"。将曝光时间设置为"200 ms"。 </li><li>在左侧面板上，点击"多维采集"，并在滤镜菜单下选择红色通道（标记为"Alexa 568"）。然后点击"时间流逝"菜单，将时间间隔设置为5分钟，持续时间为30分钟，然后单击"开始"。 注意:根据持续时间（30分钟）和流速（〜1.0μL/ min），使用30μL运行缓冲液来平衡通道内的SLB， 即所有8个通道使用240μL运行缓冲液。 </li><li> 选择"测量"选项卡下的"圆"工具，并在任意通道中绘制一个圆。选择圆圈时，右键单击，然后选择"属性"。在"配置文件"选项卡下，选中"全部T"查看荧光强度作为时间的函数。 <ol><li>在进行下一步之前，确保该曲线达到平台，表示平衡。 </li></ol></li><li>将缓冲溶液降低到与设备相等的地面以停止流动。 </li><li>保存延时文件。 </li></ol> 6.测试与PI（4,5）含P 2的SLB的PLC-δ1PH域相互作用<ol><li>使用运行缓冲液作为稀释剂准备PH域的稀释液。 注意:由于设备内有8个通道，因此至少要使用两个通道进行空白控制。选择每边的远端，并使用剩余的通道进行蛋白质稀释。测试以下PH域浓度:0.10,0.25,0.50,1.00和2.50μM。每次稀释约200μL足以在30分钟内达到平衡。 </li><li>一次一个，分离每个出口管，并将200μL的每种蛋白质稀释液加入出口通道使用移液器。不要施加任何压力，让重力做好工作。从吸液管中取出尖端并将其连接到微流体装置。 </li><li>对每个通道重复上述步骤，并确保在此过程中气泡不会引入通道。 </li><li>将进样管降低到微流体装置下方的地面，以开始将蛋白质流过微通道。将管道的自由端粘贴到废物容器。流动蛋白质稀释30分钟。 注意:这将反转流程，并允许将蛋白质引入通道。所需的平衡和体积的蛋白质稀释的时间将取决于相互作用的亲和力。 </li><li>在软件的左面板上，在"时间段"选项卡下，单击"开始"开始再次成像。 </li><li>重复步骤5.15以确保达到平衡。实验完成后，节省时间文件。 </li></ol> 7.评估膜流动性注意:荧光恢复在漂白（FRAP）实验之后，应使用每批新的SUV和清洁的玻璃盖玻片进行，以确保SLB是流体。 <ol><li>按照以前的步骤清洁玻璃盖玻片（1.1-1.6），制造微图案化PDMS块（2.1-2.5），制备SUV（3.1-3.8），组装微流体装置（4.1-4.5）和形成SLB（5.1-5.17）描述。 </li><li>将显微镜聚焦在双层上，将曝光时间设置为200 ms，并将合并为1。 </li><li>使用532 nm，2 mW激光束（13μM半径）照相漂白圆点。光漂白后立即开始在前45秒内每3秒拍摄一系列图像，然后在剩余时间内持续30秒（总持续时间15分钟）。数据采集​​完成后，保存延时文件。 </li><li>选择漂白的area使用圆形绘图工具提取荧光强度值作为时间的函数。此外，选择附近的两个区域，一个对应于未漂白区域，一个对应于没有任何SLB的区域。 </li><li>使用以下公式计算荧光恢复率（ y ）: <img alt="方程" src="/files/ftp_upload/55869/55869eq1.jpg" /> 注意:这里F t表示漂白区域的强度作为时间的函数， F i表示漂白前的荧光强度（在这种情况下使用"1"作为归一化值）; F 0是背景强度。 </li><li>绘制荧光恢复（y轴）作为时间（x轴）的函数以产生FRAP曲线。然后，将数据拟合为单个指数函数，如下所示: <img alt="方程" src="/files/ftp_upload/55869/55869eq2.jpg" /> 注意:这里A代表移动分数<em&gt; k是移动部分的动力学常数，用于计算半衰期（ t 1/2 ）: <img alt="方程" src="/files/ftp_upload/55869/55869eq3.jpg" /></li><li>使用t 1/2计算扩散常数（ D ）: <img alt="方程" src="/files/ftp_upload/55869/55869eq4.jpg" /> 注意:这里R表示激光束半径（13μm）。用于流体双层扩散常数应为≥1.0微米2 / s 以下 。 </li></ol> 8.处理数据注意:数据分析的例程将取决于显微镜，图像处理软件和正在使用的曲线拟合软件。 <ol><li>打开之前（从步骤5.17）和之后（从步骤6.6）滴定PH域的时间推移文件。转到每个时间流逝文件的最后一帧，并对所有微通道进行线扫描以获得荧光强度数据作为距离（以像素为单位）的函数（ 图6A ）。将数据传输到电子表格软件。 </li><li>对于每个微通道（PH域加法之前和之后），采样通道内的一些数据和表示基线的通道的任一侧（ 图7A ）。 注意:空白通道荧光强度应保持不变。所有其他通道被标准化为空白通道，所以至关重要的是有两个空白通道，并确保其荧光强度保持一致。 </li><li>应用下面的公式将数据规范化为空白通道;在图7B中提供了样本计算。 <img alt="方程" src="/files/ftp_upload/55869/55869eq5.jpg" /> 注意:这里ΔF 蛋白表示蛋白质通道的背景扣除荧光强度Δ; F blank表示空白通道的背景减去的荧光强度， 前和后参考蛋白质滴定步骤之前和之后， α表示校正因子，其是蛋白质通道和空白通道的荧光强度之比（[ F 蛋白 / F 空白 ] pre ）。由于荧光强度作为PH域浓度的函数而降低，归一化数据将为负值。 </li><li>使用曲线拟合软件（参见材料表 ），绘制归一化荧光作为PH域浓度的函数，并适合Langmuir等温线（ 图6C ）: <img alt="方程" src="/files/ftp_upload/55869/55869eq6.jpg" /> 注意:这里ΔF表示荧光强度相对于荧光最大变化的变化rescence强度当蛋白的饱和浓度的情况下（ΔF 最大 ），而K个D，应用程序表示表观解离常数，它等于本体蛋白质浓度（[PLC-δ1PH]），在该50％的覆盖率（膜结合复合物）。这是基于平衡的结合测量，因此归一化数据适合于简单的Langmuir等温线，以提取表观实验参数K d，app 。基于拟合软件， PLC-δ1PH-PI（4,5）P 2相互作用的K d，app为0.39±0.05μM（ 图6B ）。 </li></ol>

作者没有什么可以披露的。

每个PIP变体虽然浓度低，但存在于特定细胞器的细胞溶质表面，其中它们有助于建立独特的物理组成和细胞器膜1的功能特异性。一个的PIP的最重要的用途是作为一个特定的对接平台需要特定亚细胞定位和/或活化6,7蛋白质的众人。由于其在细胞生理学和疾病中的作用，在生理学相关的背景下研究体外蛋白质-PIP相互作用的能力是重要的。本文所述的测定方法允许在流体脂质双层的上下文中，在磷脂的混合物与极性头基的正常表现和天然长度的脂肪酰基链的情况下考虑蛋白质-PIP相互作用。 该测定结合以pH调节作为其检测系统，并且SLB作为设置在微流体平台中的模型膜系统，与其它膜结合技术相比提供了独特的优点。首先，微流体平台由于蛋白质和脂质的体积要求低（40 nL整体通道体积; 1.0 mm 2 SLB表面积），节省了材料。另外，生理学相关的脂质组合物可以被使用，同时仍保持二维膜流动性（≥1.0微米2 / s）的28，38。检测系统与ITC，SPR和具有耗散（QCM-D）监测的石英晶体微量天平相比，提供了更好的信噪比，这允许人们不仅测试蛋白质 - 膜相互作用，而且可以测试离子，小分子，肽 - 膜相互作用井19，20，39， </sup&gt; 40 。此外，pH敏感的荧光探针是高度稳定和测试19，图 20，41中的实验条件下不发生光漂白。该平台的另一个优点是能够目视观察膜表面。某些相互作用可诱导脂质微区形成和/或影响膜流动性，其可以通过光漂白（FRAP）后的荧光恢复分别直接和/或测试42 。这里描述的测定不需要超出荧光显微镜的任何昂贵的仪器。最后，最重要的是，在没有配体/受体标记的情况下评估膜相互作用使得PIP-on-a-chip测定成为优选的方法。 虽然PIP-on-a-chip测定是一种强大的技术，但是外周膜蛋白可能会不同呃在阳离子和阴离子残基的总体组成以及它们在结合位点内/周围的分布，这产生了一些挑战。一些蛋白质具有由许多碱性残基组成的PIP结合位点，而其他蛋白质仅具有少量。因此，结合位点的H + / OH -比率将不同，结合时局部pH的变化幅度也将不同。相互作用的化学计量学和PIP结合是否通过与其他脂质的相互作用而被稳定，进一步使情况复杂化。因此，特别是大蛋白质的蛋白质的pI可能不是预期荧光变化的唯一指标。虽然一些蛋白质-PIP相互作用将导致大的荧光变化，其余的可能导致小的荧光变化。在后一种情况下，可以采取至少两种措施来增强信噪比:1）在双层上使用更大的PIP水平来增加蛋白质数量recruited到表面， 即，增加招募OH的数目-离子和猝灭öSRB-POPE分子的数量; 2）增加o SRB-POPE水平，提高信噪比。 尽管目前的平台为研究外周膜蛋白提供了许多优点，但对跨膜蛋白的研究却有一些挑战。由于SLB-玻璃支撑接近（水层为约1-2纳米取决于脂质组合物），跨膜蛋白玻璃载体相互作用可促进蛋白质变性，导致功能丧失，和固定34，43，44，45 。即使更多的涉及，已经开发了各种方法来克服这些挑战，例如玻璃支撑体的表面改性以提供聚合物垫或包括脂多糖双层R内的系绳（例如聚乙二醇化脂质）以增加SLB-玻璃支撑距离34，46，47，48。 形成高质量的SLB是片上测试的最重要的方面（ 图8A ）。建议使用干净的盖玻片和新鲜的SUV以确保重复性。由于存在阴离子脂质，如PI（4,5）P 2 ，SUV是负电荷的，这降低了SUV破裂效率并影响膜流动性（ 图8B ）。因此，SUV溶液的pH调节是至关重要的质子化PI（4,5）P 2次基磷酸盐和与玻璃，以增加效率破裂49,50减少的静电排斥。不含阴离子的SUVipids，不需要任何pH调节。此外，SUV应在氧等离子体处理和接合之后注入通道，而玻璃盖玻片表面仍然是亲水性的，这是SLB形成所需的。除了SLB质量外，灰尘颗粒也是另一个问题。在器件制造过程中，捕获在通道之间的灰尘颗粒可能导致通道融合，而捕获在通道内的灰尘颗粒可能会损坏SLB和/或降低溶液流速（ 图8C， 8D ）。应定期清洁工作区域以清除灰尘。类似地，在任何步骤都应避免将气泡暴露在通道中，否则会使SLB不可逆地损坏（ 图8E ）。 规划PIP-on-a-chip测定时需要考虑的另一个参数是蛋白质储存缓冲液。如果以高浓度使用，各组分（稳定剂，还原剂，盐，抗微生物剂，螯合剂等 ）可影响荧光和/或SLB完整性。因此，应该对存储缓冲区进行滴定以测试其效果。如果观察到效果，则在滴定蛋白质之前，还应将SLB平衡至储存缓冲液条件以防止荧光漂移。考虑到这是基于pH调节的测定，如果可能，纯化的蛋白质还应该含有与运行缓冲液（在这种情况下为pH 7.0的20mM HEPES）相同的缓冲组分，以使由缓冲液失配引起的荧光漂移最小化。 总之，我们已经证明，pH调节测定可以成功地用于研究蛋白质-PIP相互作用。即使重点在于PIP，该平台可用于测试与更复杂的膜系统的相互作用，其中包括其他生理相关脂质，如磷脂酰丝氨酸NE，磷脂酰乙醇胺，磷脂酸，和胆甾醇，等等28，39，42，51。除细胞蛋白外，该测定平台对于研究人类病原体的研究平台也是有益的。例如，通过病毒和细菌编码的蛋白质已被证明与细胞膜和一些专门与PIP的52,53，54，55，56交互。因此，PIP片上测定可提供发现和表征蛋白质-PIP相互作用的小分子抑制剂的手段。

无数的相互作用和生物化学过程发生在二维流体的膜表面上。真核细胞中的膜封闭的细胞器不仅在生物化学过程及其相关的蛋白质组学中而且在其脂质组成中是独特的。一类特殊的磷脂是磷酸肌醇（PIP）。即使它们包括只有1％的细胞脂质组的，它们在信号转导，自噬和膜运输至关重要的作用，其中包括1，2，3，4。通过细胞PIP激酶肌醇头部基团的磷酸化动态产生七个PIP头基是单- ，双- ，或三-磷酸化5。另外，PIP定义膜的亚细胞同一性，并且用作含有一种或多种磷酸苷酶的蛋白质/酶的专门的膜对接位点itide结合结构域，例如，pleckstrin同源（PH），PHOX同源性（PX），和底物蛋白N端同源性（ENTH）6,7。最好研究的PIP​​结合结构域之一是在高纳摩尔 - 低微摩尔范围亲和力8内与磷脂酰肌醇4,5-二磷酸（PI（4,5）P 2 ）特异性相互作用的磷脂酶C（PLC）-δ1PH结构域，9，10，11。 已经开发了多种定性和定量的体外方法，用于研究这些相互作用的机制，热力学和特异性。在最常用的PIP结合测定中，表面等离子体共振（SPR），等温量热法（ITC），核磁共振（NMR）光谱，脂质体浮选/沉降测定和脂质印迹（脂肪印迹/ PIP条）12，13。即使这些被广泛使用，它们都具有许多缺点。例如，SPR，ITC，和NMR需要大量的样品，昂贵的仪器，和/或受过训练的人员12，13的。一些测定法形式，例如基于抗体的脂质的印迹利用的PIP的水可溶形式，并在非生理方式12，14，15，16呈现它们。此外，脂质印迹不能可靠定量，它们经常导致假阳性/阴性的观察12，17，18。为了克服这些挑战并改进当前的工具集，在上下文中基于支持的脂质双层（SLB）建立了一种新的无标记方法<html> icrofluidic平台，将其成功地应用于蛋白质-PIP相互作用的研究（ 图1）19。 用于检测蛋白质-PIP相互作用的策略是基于pH调节感测。这涉及一种pH敏感的染料，其具有直接与磷脂酰乙醇胺脂质头部组20缀合的邻 -磺酰罗丹明B（ o SRB）。 o SRB-POPE探针（ 图2A ）在低pH下是高度荧光的，在高pH下淬灭，在7.5mol％含PI（4,5）P 2的SLB中具有约6.7的pKa（ 图5B ）。 PLC-δ1PH结构域已被广泛使用，用于验证蛋白质-PIP结合方法由于其对PI（4,5）P 2（ 图5A）高特异性21,22"&gt; 23，24，25 .Hence，我们的理由是，PLC-δ1PH结构域可以用来测试其结合至PI（4,5）P 2通过PIP-上的单芯片测定法。该PH结构域构建体在此研究中使用具有净正电荷（等电点8.4），并且因此吸引OH -离子（ 图5C）在结合PI（4,5）P 2含SLBs中，PH结构域所带来的OH -离子的膜表面，这反过来又调制所述界面电势并转移öSRB-POPE（ 图5C）26，随着PH结构域浓度的函数，荧光被猝灭（ 图6A）的质子化状态。最后，将归一化的数据是适合于结合等温线以确定PH结构域-P1（4,5）P 2相互作用的亲和力（ 图6B ， 6C ）。/ P&gt; 在这项研究中，提供了详细的方案，以在微流体平台内进行蛋白质结合含PIP的SLBs。该协议使读者将微流体装置和囊泡制备装配成SLB形成和蛋白质结合。此外，提供了用于提取PLC-δ1PH域-PI（4,5）P 2相互作用的亲和度信息的数据分析方向。

<table border="0" cellpadding="0" cellspacing="0" width="1638">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Coverslip</td>
			<td style="width:156px;"></td>
			<td style="width:123px;"></td>
			<td style="width:997px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Coverslips: Rectangles</td>
			<td>Fisher Scientific</td>
			<td>12-544B</td>
			<td>22 x 40 x 0.16 - 0.19 mm, No. 1 1/2; Borosilicate Glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7X Cleaning Solution</td>
			<td>MP Biomedicals</td>
			<td>976670</td>
			<td>Detergent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PYREX Crystallizing Dish</td>
			<td>Corning</td>
			<td>3140-190</td>
			<td>Borosilicate glass dish with a flat bottom; Diameter x Height (190 x 100 mm); Distributor: VWR (89090-700)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sentry Xpress 2.0</td>
			<td>Paragon Industries</td>
			<td>SC-2</td>
			<td>Kiln</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDMS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning&#160;</td>
			<td>4019862</td>
			<td>Polydimethylsiloxane (PDMS); Distributor: Ellsworth Adhesives</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PYREX Desiccator</td>
			<td>VWR</td>
			<td>89134-402</td>
			<td>Vacuum Rated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biopsy punch</td>
			<td>Harris</td>
			<td>15110-10</td>
			<td style="width:997px;">Harris Uni-Core; 1.0 mm diameter; Miltex Biopsy Punch with Plunger (Cat. No. 15110-10) can be used as an alternative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Device</td>
			<td></td>
			<td></td>
			<td style="width:997px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma Cleaning System</td>
			<td>PlasmaEtch</td>
			<td>PE25-JW</td>
			<td>2-stage Direct Drive Oil Vacuum Pump, O2 service (Krytox Charged)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Hot Plate</td>
			<td>Benchmark</td>
			<td>H3760-H</td>
			<td>Purchased through Denville Scientific (Cat. No. 1005640)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Frosted Micro Slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>Frosted, Selected, and Precleaned; Made of Swiss Glass; Thickness: 1 mm; Dimensions: 75 x 25 mm; GR 144</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mold</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AutoCAD</td>
			<td>Autodesk</td>
			<td>v.2016</td>
			<td>Drafting software for the photomask design</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomask</td>
			<td>CAD/Art Services</td>
			<td>N/A</td>
			<td>Design with black background and clear features was printed at 20k dpi resolution on a transparent mask (5 x 7 in) by CAD/Art Services</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone Wafers</td>
			<td>University Wafer</td>
			<td>1575</td>
			<td>Prime Grade, Single Side Polished; 100 mm (4 inch) Diameter; 525 um Thickness</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 50</td>
			<td>MicroChem Corp.</td>
			<td>N/A</td>
			<td>Negative Tone Photoresist; Penn State Nanofabrication Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SU-8 Developer</td>
			<td>MicroChem Corp.</td>
			<td>N/A</td>
			<td>Penn State Nanofabrication Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SUV</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>850457C</td>
			<td>POPC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-&#945;-phosphatidylinositol-4-phosphate</td>
			<td>Avanti Polar Lipids</td>
			<td>840045X</td>
			<td>PI4P</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-&#945;-phosphatidylinositol-4,5-bisphosphate&#160;</td>
			<td>Avanti Polar Lipids</td>
			<td>840046X</td>
			<td>PI(4,5)P2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>850757C</td>
			<td>POPE; Required for the synthesis of oSRB-POPE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lissamine Rhodamine B Sulfonyl Chloride (mixed isomers)</td>
			<td>ThermoFisher Scientific</td>
			<td>L-20</td>
			<td>Required for the synthesis of oSRB-POPE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH Sensitive Fluorescent Lipid Probe (oSRB-POPE)</td>
			<td>In-house</td>
			<td>N/A</td>
			<td>In-house Synthesis (Huang D. et al. 2013)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Scintillation Vial</td>
			<td>VWR</td>
			<td>66022-065</td>
			<td>20 mL volume capacity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aquasonic 250D</td>
			<td>VWR</td>
			<td>N/A</td>
			<td>Ultrasonic Water Bath</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclepore Track-Etched Membranes</td>
			<td>Whatman</td>
			<td>110605</td>
			<td>Polycarbonate Membrane; Diameter: 25 mm; Pore Size: 0.1 um; Distributor: Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td>VWR</td>
			<td>CX1054-6</td>
			<td>HPLC grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LIPEX Extruder</td>
			<td>Transferra Nanosciences</td>
			<td>T.001</td>
			<td>LIPEX 10 mL Thermobarrel Extruder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Viscotek 802 DLS</td>
			<td>Malvern Instruments</td>
			<td>N/A</td>
			<td>Dynamic Light Scattering; Penn State X-Ray Crystallography Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>v.6</td>
			<td>Curve-fitting software for data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Axiovert 200M Epifluorescence Microscope</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AxioCam MRm Camera</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Camera</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">X-Cite 120</td>
			<td>Excelitas Technologies</td>
			<td>N/A</td>
			<td>Light Source</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa 568 Filter Set</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Ex/Em 576/603 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AxioVision LE64 v.4.9.1.0 Software</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Image Processing Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tips</td>
			<td>VWR</td>
			<td>10034-132</td>
			<td>200 uL pipette tips; Thin and smooth tip for applying the protein solution into the microfluidic channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tips</td>
			<td>VWR</td>
			<td>53509-070</td>
			<td>10 uL pipette tips; Thin and smooth tip for applying the vesicle solution into the microfluidic channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orion Star A321 pH meter</td>
			<td>Thermo Scientific</td>
			<td>STARA3210</td>
			<td>pH meter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orion micro pH probe</td>
			<td>Thermo Scientific</td>
			<td>8220BNWP</td>
			<td>micro pH probe</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-(2-Hydroxyethyl)-Piperazine-N&#39;-(2-Ethanesulfonic Acid)</td>
			<td>VWR</td>
			<td>VWRB30487</td>
			<td>HEPES, Free Acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride</td>
			<td>VWR</td>
			<td>BDH8014-2.5KGR</td>
			<td>NaCl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubing</td>
			<td>Allied Wire &#38; Cable</td>
			<td>TFT-200-24 N</td>
			<td>Internal Diameter: 0.020-0.026 inches (0.051-0.066 cm); Wall Thickness: 0.010 inches (0.025 cm); Flexible Polytetrafluoroethylene Thin-Wall Tubing; Natural Color</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nitrogen Gas - Industrial</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oxygen Gas - Industrial</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Nitrogen</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
	</tbody>
</table>

pip-on-a-chip: a label-free study of protein-phosphoinositide interactions

许多细胞蛋白质与膜表面相互作用以影响基本的细胞过程。这些相互作用可以针对膜内的特定脂质组分，如在磷酸肌醇（PIP）的情况下），以确保特定的亚细胞定位和/或活化。已广泛研究PIP和细胞PIP结合结构域以更好地了解其在细胞生理学中的作用。我们对支持的脂质双层（SLB）进行了pH调节测定，作为研究蛋白质-PIP相互作用的工具。在这些研究中，pH敏感的邻 -磺酰罗丹明B缀合的磷脂酰乙醇胺用于检测蛋白质-PIP相互作用。当蛋白质与含PIP的膜表面结合时，调节界面电位（ 即局部pH值的变化），使探针的质子化状态发生变化。通过使用磷脂酶C delta1 Pleckstr提出了pH调节测定的成功应用的案例研究在同源性（PLC-δ1PH）结构域和磷脂酰肌醇4,5-二磷酸（PI（4,5）P 2 ）相互作用为例。这种相互作用的表观解离常数（ K d，app ）为0.39±0.05μM，类似于其他人获得的K d值。如前所述，PLC-δ1PH结构域是PI（4,5）P 2特异性，显示与磷脂酰肌醇4-磷酸的结合较弱，并且不与纯磷脂酰胆碱SLB结合。 PIP片上测定优于传统的PIP结合测定，包括但不限于低样品体积和无配体/受体标记要求，测试高和低亲和力膜相互作用的能力与小和大分子，提高信噪比。因此，PIP-on-a-chip方法的使用将有助于阐明各种膜相互作用的机制。此外，这种方法可能是你的确定调节蛋白质与膜相互作用的能力的治疗剂。

我们使用pH调节测定法研究了PIP-on-a-chip微器件中的PLC-δ1PH域-PI（4,5）P 2相互作用（ 图1 ）。通过详细的方案，我们演示了如何准备和组装微流体装置组件，制备小单层囊泡（SUV）（ 图2 ），在装置内形成SLB（ 图3 ），并测试蛋白质与含有PIP的SLB的结合。 图4中描绘了典型的SLB绑定实验的流程图。作为实例，使用PH域-PI（4,5）P 2结合，在图5中示出了pH调节测定的原理。从该研究获得的结果表明，PH结构域与含PI（4,5）P 2的SLB结合。更具体地说，我们观察到，当结合时，局部pH变得更基本。这个<html> SLB中存在的o SRB-POPE荧光探针检测到局部pH变化，然后相应地淬灭（ 图6A ， 6C ）。猝灭是浓度依赖性和可饱和的，因此将数据拟合到Langmuir等温线，得到0.39±0.05μM的K d，app （ 图6B ， 6D ）。对于PI（4,5）P 2 ，PH结构域显示出选择性，因为对POPC（两性离子脂质）没有观察到结合，并且对含有PI4P的SLB（ K d，app = 1.02±0.20μM）的结合较弱（ 图6B ）。包括样品计算以显示如何处理荧光数据（ 图7 ）。在图8中 ，呈现了一系列微通道图像，以提供确定SLB和微通道质量的视觉线索。 _content"fo:keep-together.within-page ="1"&gt; <img alt="图1" class="xfigimg" src="/files/ftp_upload/55869/55869fig1.jpg" /> 图1:用于研究蛋白质-PIP相互作用的PIP-on-a-chip微流体装置。 （ A ）微流体装置具有8个微通道，8个入口和8个出口。流过彩色溶液使通道在该图像中可见。该装置的宽度（x）为2.0厘米，高度为0.5厘米（y），长度为3.0厘米（z）。 （ B ）微通道的地板是玻璃，而墙壁和天花板由聚二甲基硅氧烷（PDMS）组成。每个微通道的宽度为100μm，高度为40μm，长度为1cm。两个相邻微通道之间的间距为40μm。支撑的脂质双层（SLB）形成在玻璃表面的顶部。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55869/55869fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> </一&gt; <img alt="图2" class="xfigimg" src="/files/ftp_upload/55869/55869fig2.jpg" /> 图2.小单层囊泡（SUV）制备和质量控制研究。 （ A ）用于制造囊泡的脂质组分: 邻 -磺酰罗丹明B-POPE（ o SRB-POPE），L-α-磷脂酰肌醇-4-磷酸（PI4P），L-α-磷脂酰肌醇-4,5-二磷酸PI（4,5）P 2 ）和1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（POPC）。 （ B ）脂质囊泡的类型:SUV，大单层囊泡（LUV），巨大单层囊泡（GUV）和多层囊泡（MLV）。 SUV最适合SLB 34的制备。 （ C ）基于动态光散射（DLS）的确认小泡后脂质挤出的尺寸（通过0.1μm过滤器）。 53.9nm的流体动力学半径（Rh）证实了m<html>囊泡尺寸分布的大小约为100nm。结果表示含有PI（4,5）P 2的SUV的测量结果。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55869/55869fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55869/55869fig3.jpg" /> 图3:玻璃支架上的SLB形成。 （ A ）制备所需脂质组成的囊泡，然后流过微通道。囊泡吸附到玻璃表面并变形。一旦实现了临界表面覆盖，囊泡自发地破裂形成SLB。从点35，36改编。 （ B ）显示10X物镜下的装置内的SLB。使用Alexa 568滤波器组（576 / 603nm的激发/发射）。ource.jove.com/files/ftp_upload/55869/55869fig3large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图4" class="xfigimg" src="/files/ftp_upload/55869/55869fig4.jpg" /> 图4:典型SLB结合实验的流程图。用氧等离子体处理微流体装置部件， 即图案化的PDMS块和清洁的盖玻片，使两个表面亲水并组装。 SUV通过微通道流动以形成SLB。移除过量的囊泡，并通过流动缓冲溶液使SLB平衡至实验条件。然后，蛋白质稀释液流过微通道。最后，对荧光强度数据进行分析，归一化，并绘制为蛋白质浓度的函数。归一化数据适合提取表观离解常数（ K d，app ）va的函数略。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55869/55869fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55869/55869fig5.jpg" /> 图5:基于pH调制的传感原理。 （ A ）与PI（4,5）P 2头基肌醇1,4,5-三磷酸（Ins（1,4,5）P 3 ）（PDB）复合的PLC-δ1PH结构域的X射线晶体结构ID:1MAI） 37 。 （ B ） o SRB-POPE探针在低pH下是高度荧光的，并且在高pH下淬灭，在7.5mol％含PI（4,5）P 2的SLB中，pKa为6.7，如pH滴定曲线所示。 （ C ）本研究中使用的PH域的等电点（pI）为8.4，所以在实验pH（7.0）下，蛋白质带正电荷。带净净c哈耶，蛋白质吸引OH -离子。当PH结构域结合于PI（4,5）P 2含SLBs中，它带来的OH -离子与膜表面，界面电位被调制这继而移动öSRB-POPE的质子化状态，并且其荧光以PH域浓度依赖的方式淬灭。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55869/55869fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" class="xfigimg" src="/files/ftp_upload/55869/55869fig6v2.jpg" /> 图6:通过pH调节监测PLC-δ1PH-膜结合。 （ A ）在指定浓度下添加PH域之前和之后的微通道的视图。 （ B ）对POPC，PI4P和PI（4,5）P 2含量SLB的亲和力比较。 PH域绑定<html>至7.5mol％含PI（4,5）P 2的SLB产生K d，app为0.39±0.05μM。对于含有7.5mol％含PI4P的SLB（ K d， 1.02±0.20μM），观察到较弱的结合，并且对于纯的POPC SLB没有观察到结合。误差条表示SEM（n = 3）。使用双尾t检验来比较PI4P和PI（4,5）P 2结合之间的亲和力（* p = 0.0396）。 （ C ）通过微通道的线扫描的荧光强度被绘制为POPC，PI4P和PI（4,5）P 2结合实验的距离（以像素为单位）的函数。 （ D ）标准化和平均的POPC，PI4P和PI（4,5）P 2结合数据作为PLC-δ1PH域浓度的函数绘制，然后适合Langmuir等温线提取K d，app 。详见步骤8.4中的等式。"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图7" class="xfigimg" src="/files/ftp_upload/55869/55869fig7.jpg" /> 图7:数据处理的样本计算。 （ A ）在（黑色）和之后（红色）蛋白质滴定线扫描空白（无蛋白质）和蛋白质通道的数据之前，其中荧光强度数据作为距离（以像素为单位）的函数。阴影区域表示用于提取计算数据的区域。在每个通道之前和之后提取基线荧光强度数据。 （ B ）在蛋白质滴定之前和之后提取空白和蛋白质通道的数据。平均值用于基线和通道荧光数据。基线减法后，将荧光数据归一化为空白通道。详见步骤8.3中的等式。 <a<html> href ="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55869/55869fig7large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图8" class="xfigimg" src="/files/ftp_upload/55869/55869fig8.jpg" /> 图8:评估SLB和微通道的完整性。 （ A ）展示高品质SLB和微通道的图像。 （ B ）不完全融合。 （ C ）熔融微通道。 （ D ）捕获在微通道内的灰尘颗粒。 （ E ）微通道内的空气气泡。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55869/55869fig8large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 补充文件1:Pattern_Design.dwg空白"&gt;请点击这里下载文件。

Watch this Scientific Journal Video about PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions at JoVE.com

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

我们在微流体平台的背景下提供支持的脂质双层，以使用基于pH调节的无标记方法研究蛋白质 - 磷酸肌醇相互作用。

PIP-on-a-chip:蛋白质 - 磷酸肌醇相互作用的无标记研究

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

pip-on-chip-label-free-study-protein-phosphoinositide

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.4K 次观看.  The Pennsylvania State University. 我们在微流体平台的背景下提供支持的脂质双层，以使用基于pH调节的无标记方法研究蛋白质 - 磷酸肌醇相互作用。 

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

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

科研

生物工程

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

使用无标记光学生物传感器由G-蛋白偶联受体，以检测钾通道的调制

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics.

用无标签荧光显微镜评价活体、人体阻力动脉中胶原蛋白和弹性蛋白的压力依赖性 Microarchitectures

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...