作者非常感谢 MBI 的财政支持。

1.硅主要模具制备 注: 光掩膜是主要模具准备所需。光掩模可以从独立的供应商购买或制作内部通过标准光学掩模制作程序。明亮的领域极性光掩膜用在这种情况下 ( 图 2 )。
<ol><li>模式定义 <ol><li>自旋大衣与苏 8 3010 负性光刻 2,300 rpm，30 4 英寸硅片 s.</li>
		<li>软烤 2 分钟 65 ° C，然后在 8 分钟 95 ° C 的热板上光致抗蚀剂</li>
		<li>暴露紫外线光致抗蚀剂 (i-线，365 nm) 通过下总能量剂量的 100-120 兆焦耳/厘米 2; 光刻掩模首选硬接触模式来实现更高的分辨率。</li>
		<li>删除硅片和申请职位曝光烘烤在 65 ° C，1 分钟，然后在 2 分钟 95 ° C</li>
		<li>发展光致抗蚀剂使用苏 8 开发人员在室温，然后用异丙醇冲洗并轻轻吹干与氮; 实测厚度应当为 10 µ m 及以下。请参见 图 2 b 实际硅模具图片。</li>
	</ol></li> <li>硅烷化硅模具 <ol><li>治疗氧等离子体处理的硅胶模具为 30 60 W s 与 20 sccm 的氧流量。在过程中设置分庭的压力为 1 10 毫巴。</li>
		<li>将模具放在一个真空罐与 50 µ L 的硅烷和把罐子留在真空状态 （1-10 毫巴） 至少 2 h. 
		注: 硅烷化过程将创建疏水的表面涂层，防止粘在模具 15 聚二甲基硅氧烷。请注意，也可以使用另一种方法，涉及干法刻蚀硅的捏造主要的模具。在这种情况下，光掩模将相反极性 （暗场），并且在步骤 1.1 中的模式定义将使用正性抗蚀剂。</li>
	</ol></li></ol> 2。制备的聚二甲基硅氧烷辅助模具 <ol><li>聚二甲基硅氧烷混合 <ol><li>混合聚二甲基硅氧烷弹性体及固化剂、 10:1; 总金额是这样产生的聚二甲基硅氧烷厚度约 1 至 1.5 毫米。</li>
		<li>后彻底混合，脱气混合物由将其留在一个真空罐真空态 （1-10 毫巴），大约 15 分钟或直到有没有可见的泡沫; 这会移除所有残存的空气混合物内。</li>
	</ol></li> <li>模具复制 <ol><li>上的硅胶模具制作在步骤 1 中倒入聚二甲基硅氧烷混合和脱气的混合物用相同的设置步骤 2.1.2 删除所有残存的空气。2 h 在热板治疗混合物 70 ° C 的高温。</li>
		<li>从热板删除固化的聚二甲基硅氧烷和让它冷却到室温。用刀片，切割边缘的硅胶模具 PDMS。</li>
		<li>用一把镊子，捏紧切聚二甲基硅氧烷的一个角落，并仔细地剥掉硅模具 PDMS 副本; 由此产生的微流控模式这次要的模具是椎间盘突出症，这是相反极性的主模 （ 图 2c)。</li></ol></li> <li>硅烷化聚二甲基硅氧烷副本 （相同步骤 1.2） <ol><li>治疗与氧等离子体聚二甲基硅氧烷模具为 60 W，30 s 与 20 sccm 的氧流量。在过程中设置分庭的压力为 1 10 毫巴。</li>
		<li>模具在真空罐 50 µ L 的硅烷和至少 2 h.离开处于真空状态 （1-10 毫巴） jar 的地方</li>
	</ol></li></ol> 3。聚二甲基硅氧烷模板制备 注: 规范的形状和大小的最终设备，缓解两半的对齐方式的主要特点，采用两个单独的 PDMS 模板，其中定义几何形状的设备，透明窗口和自然出连接的位置。第一个的 PDMS 模板艾滋病的图案的一半的设备，而第二个有助于缓解平一半的设备制造制造。
<ol><li>设计模板中使用计算机辅助设计 (CAD) 软件。 图 3 显示的布局和模板用于制造该设备的图案的一半尺寸。从设计制造该设备的平一半，删除 1.5 毫米直径孔。</li>
	<li>获取模板从外部供应商或通过内部的机械车间，如果可用。 
	注: 压克力被用作模板材料由于易于制造和低成本可以实现在任何标准的厂房。有替代选项，如 3D 打印。</li>
	<li>混合的聚二甲基硅氧烷弹性体和固化剂 10:1; 一定要准备足够数量的 PDMS 完全淹没模板。</li>
	<li>后彻底混合，脱气混合物由将其留在一个真空罐真空态 （1-10 毫巴） 大约 15 分钟或直到有无可见气泡 （以较晚者为准）; 这是删除任何被困的空气混合物内。</li>
	<li>加油压克力模板的聚二甲基硅氧烷混合，直到他们最顶级的表面淹没大约 1 毫米以下的液体表面。脱气 PDMS 又要用相同的设置在 3.4 删除所有残存的空气。热这在 60 ° C 2 h 在平整的热板治疗混合物。</li>
	<li>从热板删除固化的聚二甲基硅氧烷和让它冷却到室温。用刀片，切压克力模板边缘 PDMS。</li>
	<li>用一把镊子，捏切聚二甲基硅氧烷的一个角落，仔细地剥掉压克力模板 PDMS。 
	注: 图 3 b 显示的布置和尺寸的聚二甲基硅氧烷副本用于制造该设备的图案的一半。</li>
	<li>准备第二个聚二甲基硅氧烷副本制作单位一半的设备重复步骤 3.3 至 3.7 但没有 1.5 毫米直径孔使用压克力模板。</li>
</ol> 4。微流控器件制造 <ol><li>制作的图案的一半的设备 （例如，设备布局） <ol><li>治疗与氧等离子体 CaF 2 窗口为 30 60 W s 与 20 sccm 的氧流量。这样做是为了在下面的制造过程中改善 NOA 的流量。 
		注: 此步骤不是强制。</li>
		<li>的地方仔细的第一个 PDMS 模板 （与 1.5 毫米直径支柱之一） 上平坦的表面，例如，碱石灰板 ( 图 4 )。地方在 PDMS 插头为中心的 CaF 2 窗口，然后轻轻地按窗口等，它是在与插头 ( 图 4 b) 接触良好。</li>
		<li>带 pdms 作出在第 2 步和背面的模具，与中央室 ( 图 4 的位置对齐放置紫外透明薄板 （在这种情况下，石英板、 500 μ m 厚和 1.5 厘米 × 1.5 厘米大小）c).确保石英板是在与 PDMS 模具接触良好。 
		注: Th电子石英板防止模具不需要的地区很容易接触到 CaF 2 窗口。</li>
		<li>轻轻地点与 CaF 2 窗口的中心对齐，射流分庭向 CaF 2 窗口这 PDMS 模具面朝下。确保所有的元素 （模板、 模具和窗口） 能良好的接触，并对齐 ( 图 4 c-4 d)。</li>
		<li>逐步免除诺亚在 PDMS 模板中让滴，让它慢慢填充型腔。一旦树脂接触到窗口的边缘，毛细管流将之间的差距薄 (~ 10 µ m) 的聚二甲基硅氧烷模具和 CaF 2 窗口 ( 图 4 e 4f)。</li>
		<li>后腔完全填满，治愈 NOA 暴露于 UV 光 （例如，与紫外 LED 曝光系统， 图 4 g）。 
		注: 曝光时间可能会发生变化因此 UV 光源的能量。紫外 LED 曝光系统，提供 24 兆瓦/厘米 2 功率密度，需要大约 90 100%的动力，连续曝光模式 s.</li>
		<li>小心地从后面的聚二甲基硅氧烷模具卸下薄石英板，然后轻轻剥离 PDMS 模具从诺亚层 ( 图 4 h) 的顶部。最后，从 PDMS 模板 ( 图 4 我) 中删除 NOA 层。 
		注意: 生成的设备布局上固化 NOA 会有模式的相同的极性，在初生硅模具。</li>
	</ol></li> <li>制作设备 （即，如果没有设备的布局） 平半 <ol><li>对待 CaF 2 窗口与氧等离子体为 30 60 W s 与氧流量 20 sccm。 
		注: 此步骤不是强制。</li>
		<li>仔细地放置在平坦的表面，例如，碱石灰板上第二个的 PDMS 模板 （即没有 1.5 毫米直径支柱）。地方为中心在 PDMS 插头的 CaF 2 窗口，然后轻轻地按窗口等，它是在与插头接触良好。</li>
		<li>地方 1 毫米厚的聚二甲基硅氧烷板 5 厘米 x 3.5 厘米大小 CaF 2 窗口的顶部与聚二甲基硅氧烷板与 PDMS 模板的中心对齐。聚二甲基硅氧烷板是否与窗口接触良好。</li>
		<li>逐步免除诺亚在 PDMS 模板中让滴，让它慢慢地充满腔。</li>
		<li>后腔完全填满，治愈 NOA 暴露于 UV 光 （例如，与紫外 LED 曝光系统）。 
		注: 曝光时间可能会发生变化相应地与 UV 能量来源。用紫外 LED 曝光系统，提供 24 兆瓦/厘米 2 功率密度，这就需要大约 50 100%的动力，连续曝光模式 s.</li>
		<li>皮诺亚层自上而下的聚二甲基硅氧烷表并小心地从 PDMS 模板卸下 NOA 层固化。</li>
	</ol></li> <li>粘接的两半的设备 <ol><li>这样两个 CaF 2 窗口对齐对齐两半的设备。轻轻地手指压角的诺亚层左右两边这样两个半场的位置固定的。</li>
		<li>切出 1 毫米厚的聚二甲基硅氧烷板使用 8 毫米直径的打孔器 ( 图 5 ) 的两个圆形光盘。</li>
		<li>从 1 毫米厚的聚二甲基硅氧烷钢板切割具有相同大小的设备 （4 厘米 × 2.5 厘米） 的两个矩形。在这两个聚二甲基硅氧烷的矩形，削减开口对应频道和中-让/出-自然的设备. 
		注: 在 PDMS 矩形的预切的开口注定要从倒塌压过程中防止渠道。</li>
		从底部的 <li>堆栈在以下顺序: 一聚二甲基硅氧烷矩形预切开口，一个聚二甲基硅氧烷光盘 （接触底部窗口，插嘴步 4.3.2），手指按下两个半场设备，第二次的聚二甲基硅氧烷光盘 （坐在顶部窗口），和最后预切开口 ( 图 5 b) 第二个聚二甲基硅氧烷矩形。</li>
		<li>将此程序集放在真空压安装，这样它夹在 2 片和密封的塑料袋 ( 图 5 c)。开启真空泵和疏散大会。让真空泵达到其底部的压力或申请至少 10 分钟真空 
		注: 底部压力实现取决于真空泵使用和塑料袋的封口质量。</li>
		<li>暴露紫外线用宽频带汞气灯在 270 15 分钟，轮到 W 关闭真空泵，让大会慢慢地发泄到大气压力之前删除从大会的最后设备撤离的大会。</li>
	</ol>
	</li>
</ol>

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作者没有透露。

为了评估和优化制造协议，我们使用一个简单的布局，为微流控模式与大型矩形室 （5 毫米 x 2.5 毫米大小） 的中心，两个小矩形分庭 （5.5 毫米 x 0.75 毫米大小） 分开的主电路上上部和下部的边和 300 微米宽的-让/出-自然渠道。中央墓室用于播种和细胞，而两个分离小分庭用于测量大气的背景在红外光谱中观察实验作为参考室，如以前的出版物13所述。在让和外让中央室连接到?...

傅里叶变换红外分光显微镜 (FTIR) 已被广泛用作标签免费和非侵入性的成像技术，提供详细的化学信息的样本。这使提取的生化信息学习化学的生物样品，用最少的准备因为吸收谱的标本进行的内在指纹图谱及其化学成分1,2.最近，FTIR 已越来越多地应用于活的生物样品，例如，单元格3的研究。然而，水，这是在大多数情况下活细胞中，在中红外区域显示了较强的吸收。即使作为一个薄层，它的存在能够完全战胜标本的重要结构信息。
多年来，常用的方法是固定或干燥样品，完全排除水吸收信号的频谱。然而，这种方法允许不为活细胞的实时测量的研究是十分必要的化学成分及其细胞过程随时间变化。从活的生物样品，获得可靠的吸收谱的一种方法是限制到小于 10 µ m4红外梁在介质中的光学路径总长度。
在生活中的既定方法细胞实验一直到目前为止，减毒总反射 (ATR)-红外光谱成像，使测量的样品厚度，让细胞持续在水介质中厚层独立。然而，倏逝波的小深度限制样品测量从表面的 ATR 水晶5只第一的几个微米。
另外，水吸收限制已被规避与各种微流控系统，通常可以分为两大组的出现: 打开的通道 （在流体表面之一暴露在大气之中），并关闭通道 （其中两个红外透明窗口以进行分隔间隔定义的厚度）。
Loutherback等人开发开放通道膜装置，使活细胞达 7 天6长期限连续红外的测量。该方法需要高湿度的环境，以防止介质从细胞表面蒸发。该系统最适合与自然生长在气-液界面，如皮肤、 肺和眼睛或微生物的生物膜7上皮组织的细胞。
关闭通道配置旨在创建一个统一、 薄层之间两个平行的红外透明窗口，而将细胞保持其水介质中。这腔厚度是这样水吸收信号低于饱和。然后可以减去水背景以获取正确的样品光谱。大部分的封闭通道方法利用塑料间隔分离这两个窗口，形成一个可拆卸的液体室3，8，9。此方法的一个优点是它不需要微细加工;然而，比自然出渠道测量室更复杂的结构都是极难实现的薄垫片。也是由于其依赖对机械夹紧的红外测量之间的路径长度的重现性问题。为了实现更可靠的光谱采集的间距更精确的控制，光刻方法，已实施模式光致抗蚀剂在红外衬底来定义间隔9，10,11,12.尽管这使得更复杂的结构，在间隔中定义，该方法需要对微细加工工厂生产模式在每一个衬底上的访问。
在本文，我们提出一种红外兼容微流控装置，简单的制作技术与目标，降低制造成本和访问微细加工设施的要求。提出的方法在这里 （请参见图 1） 使用既定的过程称为软光刻技术。在这种情况下，两个模具是必需的。主要的模具是由 4 英寸硅片，使用标准的 UV 光刻工艺。二级模具是由聚二甲基硅氧烷，这在硅主要模具有极性的模式，并作为后续设备制造的模具师傅其副本。
该装置具有两个单独的图层: 第一层与微流控布局 （在介绍案件和组成的微流控通道，在-/ 出放开，CaF2视口观察室） 和一个平坦的表面 （第二层其中包括只 CaF2视区）。
在这里 UV 固化的光学胶，诺兰光学粘接 73 (NOA73，简称 NOA 从此)，用于塑料体主要的设备。有使用此光学胶粘剂的几个优点: 低制造成本、 易用性与外部系统、 光学透明度好、 低粘度和最重要的是，生物相容性13的连接。CaF2作为其生物相容性和优良的红外透明14视区是一个合适的选择。
这种新的方法，对微细加工设施的访问是严格要求只为主要模具制造。塑料微流控装置随后制备工艺可以在任何实验室配有 UV 光源进行。

<table><tbody><tr><td>&lt;strong&gt;Chemical&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane 97%</td><td>Sigma Aldrich</td><td>448931-10G</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td></td><td>Polydimethylsiloxane or in short, PDMS</td></tr><tr><td>Norland Optical Adhesive 73</td><td>Norland Products Inc.</td><td>7304</td><td></td></tr><tr><td>SU8 3010 photoresist</td><td>MicroChem</td><td>Y311060</td><td></td></tr><tr><td>SU8 developer</td><td>MicroChem</td><td>Y020100</td><td></td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Silicon wafer, 4 inch, prime grade</td><td>Bonda Technology Pte Ltd</td><td></td><td></td></tr><tr><td>CaF2 IR-grade windows</td><td>Crystran, UK</td><td>CAFP10-1</td><td>10 mm diameter, 1 mm thickness</td></tr><tr><td>Acrylic templates</td><td></td><td></td><td>Custom made</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>UV-KUB 2 (UV LED exposure system)</td><td>KLOE</td><td></td><td>Emission spectrum 365nm &amp;plusmn; 5nm</td></tr><tr><td>Newport UV lamp</td><td>Newport</td><td>Model 66902</td><td>50-500 Watt Hg arc lamp</td></tr><tr><td>CEE Spin coater</td><td>Brewer Science</td><td>Model 200x</td><td></td></tr><tr><td>MJB4 mask aligner</td><td>SUSS MicroTec</td><td></td><td></td></tr><tr><td>Precision digital hot plate</td><td>Harry Gestigkeit GmbH</td><td>2860SR</td><td></td></tr><tr><td>Plasma Surface Technology</td><td>Diener Electronic GmbH + Co. KG</td><td></td><td>For O&lt;sub&gt;2&lt;/sub&gt; plasma treatment</td></tr><tr><td>IDP-3 Dry Scroll Vacuum Pump</td><td>Agilent Technologies</td><td></td><td>ultimate pressure 3.3 x 10&lt;sup&gt;-1&lt;/sup&gt; mbar</td></tr><tr><td>Bruker IFS 66v/s FTIR Spectrometer</td><td>Bruker</td><td></td><td></td></tr></tbody></table>

soft lithographic procedure for producing plastic microfluidic devices with view-ports transparent to visible and infrared light

红外 (IR) 分光显微镜的生活生物样品被阻碍水吸收的中红外范围内，适用于微流控设备缺乏。在这里，表现出塑料微流控器件的制备一种协议，软光刻技术用于嵌入透明氟化钙 (CaF2) 查看端口与观察理事会。该方法基于一个副本铸造方法，聚二甲基硅氧烷 (PDMS) 模具制作标准平版印刷手续，然后作为模板用于生产塑料设备。该塑料器件具有紫外/可见光和红外 (UV/Vis/IR)-透明窗口由 CaF2 ，以便直接观察可见和红外灯。该方法的优点包括: 减少对塑料的身体，灵活的设计，通过访问一个洁净室微加工设施，多个视图端口，方便和灵活的连接到外部的抽油系统的需求如，打开/关闭通道配置和添加复杂的功能，如纳米多孔膜的可能性。

图 6显示了一个全新的 CaF2窗口，图案半数的设备和完整的设备的透过率光谱。所有三种光谱展现优秀透明度中期 IR 透光率大于 80%。可见光谱中完整的器件 （图中黄色曲线） 的干涉图样被造成空气隙在 9-10 µ m 两扇窗户之间的范围。这些光谱表明，这里提出的制备方法并不改变 CaF2中期的红外光谱范围内的透明度。
<p class="jove_cont...

Watch this Scientific Journal Video about Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light at JoVE.com

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

该协议为塑料微流控器件的制备与透明查看端口的可见光和红外线光成像的描述。

生产塑料微流控装置与查看端口对可见光和红外透明软光刻过程

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...

soft-lithographic-procedure-for-producing-plastic-microfluidic

Research

JoVE Journal

Bioengineering

13.0K 次观看.  National University of Singapore. 该协议为塑料微流控器件的制备与透明查看端口的可见光和红外线光成像的描述。

视频: Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

微流体技术，探讨细胞变形

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

科研

生物工程

One-Step Approach to Fabricating Polydimethylsiloxane Microfluidic Channels of Different Geometric Sections by Sequential Wet Etching Processes

Polydimethylsiloxane (PDMS) materials are substantially exploited to fabricate microfluidic devices by using soft lithography replica molding techniques. Customized channel layout designs are necessary for specific functions and integrated performance of microfluidic devices in numerous biomedical and chemical applications (e.g., cell culture, biosensing, chemical synthesis, and liquid handling). Owing to the nature of molding approaches using silicon wafers with photoresist layers patterned by photolithography as master molds, the microfluidic channels commonly have regular cross sections of rectangular shapes with identical heights. Typically, channels with multiple heights or different geometric sections are designed to possess particular functions and to perform in various microfluidic applications (e.g., hydrophoresis is used for sorting particles and in continuous flows for separating blood cells6,7,8,9). Therefore, a great deal of effort has been made in constructing channels with various sections through multiple-step approaches like photolithography using several photoresist layers and assembly of different PDMS thin sheets. Nevertheless, such multiple-step approaches usually involve tedious procedures and extensive instrumentation. Furthermore, the fabricated devices may not perform consistently and the resulted experimental data may be unpredictable. Here, a one-step approach is developed for the straightforward fabrication of microfluidic channels with different geometric cross sections through PDMS sequential wet etching processes, that introduces etchant into channels of planned single-layer layouts embedded in PDMS materials. Compared to the existing methods for manufacturing PDMS microfluidic channels with different geometries, the developed one-step approach can significantly simplify the process to fabricate channels with non-rectangular sections or various heights. Consequently, the technique is a way of constructing complex microfluidic channels, which provides a fabrication solution for the advancement of innovative microfluidic systems.

Several methods are available for the fabrication of channels of non-rectangular sections embedded in polydimethylsiloxane microfluidic devices. Most of them involve multistep manufacturing and extensive alignment. In this paper, a one-step approach is reported for fabricating microfluidic channels of different geometric cross sections by polydimethylsiloxane sequential wet etching.

连续湿法刻蚀法制作不同几何断面烷微流控通道的一步法

Polydimethylsiloxane (PDMS) materials are substantially exploited to fabricate microfluidic devices by using soft lithography replica molding techniques. ...

工程学

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

研究多孔介质中生物堵塞的微流体平台

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

环境科学

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Fabrication Techniques for High-Pressure Testing of Microscale Supercritical CO2 Foam Transport in Fractured Unconventional Reservoirs

Pressure limitations of many microfluidic platforms have been a significant challenge in microfluidic experimental studies of fractured media. As a result, these platforms have not been fully exploited for direct observation of high-pressure transport in fractures. This work introduces microfluidic platforms that enable direct observation of multiphase flow in devices featuring surrogate permeable media and fractured systems. Such platforms provide a pathway to address important and timely questions such as those related to CO2 capture, utilization and storage. This work provides a detailed description of the fabrication techniques and an experimental setup that may serve to analyze the behavior of supercritical CO2 (scCO2) foam, its structure and stability. Such studies provide important insights regarding enhanced oil recovery processes and the role of hydraulic fractures in resource recovery from unconventional reservoirs. This work presents a comparative study of microfluidic devices developed using two different techniques: photolithography/wet-etching/thermal-bonding versus Selective Laser-induced Etching. Both techniques result in devices that are chemically and physically resistant and tolerant of high pressure and temperature conditions that correspond to subsurface systems of interest. Both techniques provide pathways to high-precision etched microchannels and capable lab-on-chip devices. Photolithography/wet-etching, however, enables fabrication of complex channel networks with complex geometries, which would be a challenging task for laser etching techniques. This work summarizes a step-by-step photolithography, wet-etching and glass thermal-bonding protocol and, presents representative observations of foam transport with relevance to oil recovery from unconventional tight and shale formations. Finally, this work describes the use of a high-resolution monochromatic sensor to observe scCO2 foam behavior where the entirety of the permeable medium is observed simultaneously while preserving the resolution needed to resolve features as small as 10 &#181;m.

Microfluidic Fabrication Techniques for High-Pressure Testing of Microscale Supercritical CO2 Foam Transport in Fractured Unconventional Reservoirs

This paper describes a protocol along with a comparative study of two microfluidic fabrication techniques, namely photolithography/wet-etching/thermal-bonding and Selective Laser-induced Etching (SLE), that are suitable for high-pressure conditions. These techniques constitute enabling platforms for direct observation of fluid flow in surrogate permeable media and fractured systems under reservoir conditions.

破碎非常规储层微尺度超临界CO泡沫运输的高压 试验微流体制造技术

Pressure limitations of many microfluidic platforms have been a significant challenge in microfluidic experimental studies of fractured media. As a ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

窗口上的一个微观世界：简单的微流体系统为研究多孔介质中的微生物运输

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

生物学

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

多级可变高度的光刻多层带瓣微流体装置

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

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

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

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

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

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

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

用于研究和教育应用的定制微流体器件的快速制造

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

用于组合插头生产的微流体装置的设置

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

作者聚焦：在精准肿瘤学中整合计算和实验方法

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

用于组合塞生产的双层微流控装置

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...

生产塑料微流控装置与查看端口对可见光和红外透明软光刻过程

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摘要

探索更多视频

此视频中的章节

微流体技术，探讨细胞变形

连续湿法刻蚀法制作不同几何断面烷微流控通道的一步法

研究多孔介质中生物堵塞的微流体平台

高通量单细胞的分离和文化的微流体平台

破碎非常规储层微尺度超临界CO泡沫运输的高压 试验微流体制造技术

窗口上的一个微观世界：简单的微流体系统为研究多孔介质中的微生物运输

多级可变高度的光刻多层带瓣微流体装置

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

用于研究和教育应用的定制微流体器件的快速制造

用于组合塞生产的双层微流控装置

破碎非常规储层微尺度超临界CO泡沫运输的_高压试验微流体制造技术