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。

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			<td height="42" style="height:42px;width:291px;">Trichloro(1H,1H,2H,2H-perfluorooctyl)silane 97%</td>
			<td style="width:212px;">Sigma Aldrich</td>
			<td style="width:112px;">448931-10G</td>
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			<td height="42" style="height:42px;width:291px;">Sylgard 184 Silicone Elastomer Kit</td>
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			<td style="width:243px;">Polydimethylsiloxane or in short, PDMS</td>
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			<td height="21" style="height:21px;width:291px;">Silicon wafer, 4 inch, prime grade</td>
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			<td height="21" style="height:21px;width:291px;">CaF2 IR-grade windows</td>
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			<td style="width:243px;">Emission spectrum 365nm &#177; 5nm</td>
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			<td height="21" style="height:21px;width:291px;">Newport UV lamp</td>
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			<td height="21" style="height:21px;width:291px;">Precision digital hot plate</td>
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			<td style="width:112px;">2860SR</td>
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			<td height="42" style="height:42px;width:291px;">Plasma Surface Technology</td>
			<td style="width:212px;">Diener Electronic GmbH + Co. KG</td>
			<td style="width:112px;"></td>
			<td style="width:243px;">For O2 plasma treatment</td>
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		<tr height="23">
			<td height="23" style="height:23px;width:291px;">IDP-3 Dry Scroll Vacuum Pump</td>
			<td style="width:212px;">Agilent Technologies</td>
			<td></td>
			<td>ultimate pressure 3.3 x 10-1 mbar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Bruker IFS 66v/s FTIR Spectrometer</td>
			<td style="width:212px;">Bruker</td>
			<td></td>
			<td></td>
		</tr>
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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

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

软平版功能化和图案无氧化硅和锗

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

科研

生物工程

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

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 ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

微流控生物工程血管化组织和细胞器解体

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

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 ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

生物灵感软机器人与合并微电极

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...