This work was supported by Tufts University and by a generous gift from Dr. James Kanagy. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. (DGE-1325256) that was awarded to S.C.F. D.J.W. was supported by a U.S. Department of Education GAANN fellowship. We thank Dr. Jeremy Schonhorn (JanaCare), Dr. Jason Rolland (Carbon3D), and Rachel Deraney (Brown University) for helping develop the design of the three-dimensional paper-based microfluidic device and immunoassay.

1.基于纸张的微流体器件层的制备<ol><li>准备用于使用图形设计软件程序的纸张，尼龙，和粘合剂层的图案。 6每个层可以具有不同的图案。 注:该图案可以包括被不需要的官能纸质免疫对准孔，但与三维器件的重现性制造协助。如果设备是单独组装，成条，或作为全片这些孔的位置会有所不同。用于设计图形的软件程序可能基于图案化技术（ 例如 ，光刻，蜡染，或切割）的选择。 6 </li><li>用的70％（体积/体积）乙醇和水的溶液喷雾的工作区。用干净纸巾的工作区。 </li></ol> 2.纸层的制备:样品增量，共轭寄存，孵化，洗层<ol><li>准备使用大型台式切纸机定性滤纸的层。切股票纸上成标准的纸张尺寸使用固体油墨（蜡）打印机，以方便构图。例如，一个460点¯x570毫米2片可以让4张美书纸（8.5 x 11英寸2）。在任何时候都处理好与干净的手套纸，以减少污染。 </li><li>装入层析纸的单页纸进打印机纸盒。打印先前设计层（参见图1）。 注:图案可以直接印刷在使用自动进这个表。只有一张纸应在同一时间内打印，以避免卡纸。对于所有图层，使用"增强"打印设置。 </li></ol> 3.尼龙薄膜层的制备:捕获层<ol><li>削减库存辊尼龙膜的成用台式裁纸刀片（7.5×10英寸2）。在处理尼龙采取非常谨慎，膜，以保持它的完整性，并防止撕裂。存放在干燥器柜未使用的材料，如尼龙膜是湿气敏感。 注:单页纸比美国Letter纸张窄。因为尼龙膜是薄且脆弱的，它们不能由打印机直接处理和要求的支持。细节将在下面讨论。 </li><li>使用蜡打印机，打印捕获层图案到一张复印纸，并将其粘在光盒以作为尼龙膜的定位的导向。光盒有助于多个层的对准。 </li><li>将复印纸丢球到复印纸的先前打印纸。胶带纸灯箱丢球，但不要带两片在一起。 </li><li>尼龙膜的裁切纸放置到一块干净的复印纸。确保膜覆盖复印纸的底层的印刷区域。用胶带将尼龙膜四边的丢球的复印纸。 注意:确保尼龙膜的平整，光滑，以便有与印刷（ 如卡纸或蜡的印刷不均匀），没有任何问题。蜡可以其中尼龙膜附连到复印纸在磁带上进行打印。如果发生这种情况，由于胶带覆盖，其中的尼龙是不完全的图案化的区域应被丢弃。对于未来的制剂，较大的尼龙膜件可以用来避免这种打印错误。 </li><li>装入尼龙膜（贴吧复印纸支持）的薄片装入手动进纸打印机纸盒。在同一时间只能打印一张尼龙膜。 注:对于印迹层不需要准备步骤，因为它是没有图案。 </li></ol> 4.创建印刷层的疏水性障碍<ol><li>胶带印刷层到用于放置在重力对流烘箱时甚至上方和层下加热丙烯酸类帧。请贴在尼龙膜复印纸的支撑片，直到蜡之后被熔化并形成疏水屏障。 注:丙烯酸帧是一个特制的，激光切割件的1/2。"正在制作中使用依赖于设备的数量厚的丙烯酸塑料两帧尺寸较小的框架外的边境措施11 5/8" ×2 3/4"，帧的内孔测量10 3/8"×1 3/4"。所述更大的帧的外边缘测量11 5/8"×8 7/8"，和内框架的孔测量10 1/4"×7 7/8"，开放式，内部空间允许甚至通过纸的整个厚度熔化蜡。 </li><li>放置在烤箱的层在150℃，30秒，直到蜡熔化到纸的厚度。确认该蜡已通过翻转，并检查在设计缺陷渗透的纸的厚度。 注:强制空气炉或热板，也可以使用以熔化固体蜡油墨。熔倍或温度可能取决于加热方法而变化。 </li><li>从亚克力相架取出纸张和尼龙膜。此外，从复印纸的支持纸取出尼龙膜。 </li></ol> 5.准备粘合层的<ol><li>先前准备使用机器人的刀绘图仪胶膜，用设计文件的模式双面纸（步骤1.1）。使用保护蜡衬垫纸外露粘合剂表面。 注:双面粘合剂应当具有孔，其允许样品通过层作为连续流体通路流来图案化。蜡衬垫易于从粘合剂去除，并用于在切割过程中，以保护它免受污染和撕裂。激光切割机或模具压也可被用来粘接膜的图案层。 </li></ol> 6.背衬胶粘剂器件层<ol><li>喷雾用的​​70％（体积/体积）乙醇和水的溶液中的光盒。用干净的纸巾擦拭ŧowel。 </li><li>胶带纸或尼龙膜图案层，需要用粘合剂备份到与印刷面朝下的灯箱。 </li><li>果皮从粘合剂的图案化片材保护性衬里的一侧，它固定到纸或尼龙膜上的层。使用灯箱，确保图案正确对齐。按在一起。放置部分组装设备进入一个保护滑移。 注:保护滑是通过确保他们不接触层压辊免受污染或损坏设备层压薄膜后盾一张折叠。 </li><li>通过将得到的两层组件通过自动层压到完全按下粘合剂和纸张一起从邻接层除去空气中的任口袋。 注:设备层之间的气穴可以与设备的完整性，并导致泄漏排汗重复性干扰。 </li></ol> 7.共轭升治疗艾尔与试剂免疫此前器件大会<ol><li>胶带缀合物层上的丙烯酸类帧，使得要被处理的亲水性区域与框架接触暂停，而不是。 </li><li>在1×磷酸盐缓冲盐水（PBS）加入2.5微升100毫克/毫升牛血清白蛋白（BSA）与缀合物层上的亲水区。允许其在室温下干燥2分钟，然后在65℃下5分钟。 注意:此卷是刚好足以润湿纸的区域。的BSA溶液有助于防止在干燥过程中的胶态纳米颗粒的聚集，这将有利于纳米颗粒的释放时的纸张和试剂通过样品再水化。 </li><li>添加5微升5外径胶体金纳米颗粒缀合到抗β-hCG的抗体，并重复干燥过程。 注:胶态金纳米颗粒的浓度的单位通常表示为如通过ABSO测量的光密度（OD）rbance在λ= 540纳米。没有治疗需要在第10装置装配前灯芯垫。 </li></ol> 8.横向通道的治疗试剂免疫此前器件大会<ol><li>胶带横向沟道层上的丙烯酸类帧，使得要被处理的亲水性区域与框架接触暂停，而不是。 </li><li>添加10微升阻断剂的（5毫克/毫升脱脂奶粉和0.1％（体积/体积）Tween 20的1×PBS中）来治疗的横向通道。重复相同的干燥处理（在室温下2分钟，然后在65℃，5分钟）作为共轭层。 </li></ol> 9.试剂免疫此前器件大会捕获层的治疗<ol><li>胶带捕获层上的丙烯酸类帧，使得要被处理的亲水性区域与框架接触暂停，而不是。 </li><li>以1毫克/毫升的抗α-hCG的抗体5μL的治疗捕获层然后允许样品在室温下干燥2分钟，接着8分钟，在65℃。 </li><li>添加阻断剂的2液（5毫克/毫升脱脂奶粉和0.1％（体积/体积）Tween 20的1×PBS）中。重复干燥过程的捕获层。 注:该量是合适的涂层不阻断尼龙膜，也使用多阻断剂时，可发生的孔中的论文。 </li></ol>基于纸张的10.大会三维微流体装置<ol><li>大盘洗层灯箱（朝上打印面）。如果使用的定位孔，使用手持打孔工具后续层将其删除。 </li><li>在捕获层的背面的保护膜，露出粘性。对齐用的定位孔作为引导洗涤层之上的捕获层。按两层在一起。避免接触的亲水区，以尽量减少污染或损坏设备。镊子可以用于协助assembLY。 </li><li>在孵化层的背面去除保护膜以暴露出粘合剂。对齐捕获层之上的孵化层，然后按在一起。继续以这种方式添加的层，直到所有的有源层被组装。 </li><li>放置部分组装设备插入保护滑，坚决加盖层使用层压在一起。 </li><li>在洗涤层的背面去除保护膜和印迹层固定到该装置的底部。重复层叠步骤10.4完成三维纸基微流体装置的组装。切期望数目从条带或用剪刀完全组装设备的薄片的设备。 注:设备的完整片材，设备的条带，或单一的装置可使用类似的方法来制备。 </li></ol> 11.执行基于纸张的免疫<ol><li>加入20μl的样品的亲水区在装置的顶部（ 即 ，叔他品尝层）。 </li><li>等待样品完全芯吸进入设备，则（在1×磷酸盐缓冲盐水0.05％体积/体积的吐温20）中添加15微升洗涤缓冲液中。洗涤缓冲液的第一等分试样完全邪恶到设备后，加入洗涤缓冲液的第二个15微升等分试样。 注意:洗涤缓冲液具有完全恶成当液滴已经消失，显示在纸张的表面上没有凹凸的设备。该试验完成时，洗涤缓冲液的第二个等分试样已经完全进入该设备。 </li><li> 揭示该测定的结果，使用镊子以暴露捕获层剥离设备的三个顶层。 <ol><li>定性通过观察颜色的存在或不存在解释该测定的结果。可替代地，图像用台式扫描仪和使用图像处理软件或算法来量化结果和内表征强度的分布的读出层检测区域。 20 </li></ol></li></ol>

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The authors have nothing to disclose.

确定一个可重复的制造策略是试验发展的一个重要组成部分。 22我们使用顺序，层-层的方法来制造三维纸基微流体装置。相反那些适用折叠或折纸技术来产生从纸23的单个片材的多层器件的方法，24添加剂制造提供了许多优点:（ⅰ）多材料可以并入到单个设备架构无需修改方法印刷，对齐，或层的装配。 （二）图案化粘合膜可以集成到组装过程。这些膜粘贴相邻层，并且，基于所述粘合剂的强度，可以是可逆的，以使剥离和内部层的评估。此外，粘合剂提供结构完整性的3维器件，这就排除了需要为粘结剂25剪辑或加工的外壳。 23（ⅲ）美国信纸色谱纸的各个片材可以容纳重复的阵列，从而可大大提高实验室规模制造的吞吐量（ 图5）。评估需要技术重复无数实验条件时，这是特别有益的。通过这种方式，70三维纸基装置可以同时制备。 （ⅳ）类似的多层层压方法被用于众多的商业产品的高产量制造医疗（ 例如 ，伤口护理敷料和透皮贴剂），这因此降低了生产障碍翻译这些三维纸基微流体装置。 除了便于剥离和装配，粘合剂的选择也与三维流体网络的设计是至关重要的。一个广告hesive膜可以作为纸层之间的附加屏障，它可以使上相邻的层亲水区的掩蔽。在实践中，使用的粘合剂的薄层是可取的。如果粘合剂太厚（ 例如 ，许多双面胶带），那么的纸层之间形成的间隙将太大，以促进芯吸，并且必须以亲水性物质（ 例如 ，纤维素粉）填充重获功能。 12虽然这额外的步骤增加了复杂性的制造和使用可能会与某些试验干扰物质，这些差距成为生产控制，流体下推阀的一个有用的功能。 15粘合剂的其他形式已在三维纸基微流体装置的制造中被使用。粘合剂喷雾剂提供一种简单的方法来固定层彼此。使用这种方法26，粘合剂材料被均匀地施加到两者是叔他的纸的疏水和亲水区。这种方法的优点在于，不需要额外的设备（ 例如 ，刀绘图仪或激光切割）来设计用于粘合层的图案。然而，对于粘合剂喷雾的均匀应用的条件，必须通过实验所用材料的每个类型确定。该材料的形貌可能影响粘合剂材料界面和可能需要的粗糙表面更长喷雾倍。此外，喷洒粘合剂到所述流体路径的亲水区可以通过改变纸张的润湿性导致受损的芯吸。可替代地，使用网板27或丝网印刷8的可用于图案粘合剂直接在图案化的纸层。 用于三维纸基微流体装置的发展的两个主要因素是材料的选择和flui的设计DIC网络。 （a）我们选择基于芯吸率，湿强度，厚度，和蛋白质结合能力的材料和材料的组合。芯吸速率可以影响的测定法的持续时间和时间的试剂的量有反应或一个层内结合。不同等级的纸张的特征在于毛细基于费率，例如纸，其孔隙率，其厚度的治疗。它可使用的纸张的多个层以增加器件的有效芯吸速率。 28良好的湿强度是希望的对于需要处理（ 如 ，剥落的免疫测定法）的设备已经饱和与样品后的应用程序。太厚或材料不能通过商业打印机传递由于脆弱，需要另一种方法，以产生图案化的信道（ 例如 ，光刻法）。然而，与此相反，较厚的材料是理想的印迹层（或汇）通过第绘制流体E系列FPGA芯片。尼龙膜的许多等级是市售的，其可在其不可逆地结合的蛋白质的捕获区的能力有所不同。材料取代（ 例如 ，硝化纤维素代替尼龙膜）也可影响结合能力，这可能会影响测定的灵敏度。 （二）在流体网络的设计中使用的对称性确保构图成三维器件的唯一通道的行为相同（ 例如 ，同时填充），这是对于多重分析至关重要。 19对称性进一步简化了分层设计，装配设备的整页时层对齐助攻，并能最大限度地减少浪费。修改到装置的设计会影响测定的性能。例如，增加在孵化层的横向通道的长度将影响测定的持续时间，因为流体将到达在ou之前灯芯一个按比例更长的距离tlet。 17在依赖于结合的靶生物分子的应用中，较长的检测时间可以是有利的，因为它可以提高结合的，标记物的级分之前固定所述捕获层上。 总之，我们已经提出了制造支持免疫测定的开发三维纸基微流体装置的方法。此方法，它使用一种类型的添加剂制造的，以产生多层器件，有利于生产设备中的一个规模适合于实验室研究。在这个手稿中描述的协议是专用于纸质免疫测定装置;然而，我们预期有关，这些免疫蜡印的装配程序，图形化胶，定向膜和层压，将很容易扩展到众多的三维纸基微流体器件结构。制造方法的理解会导致到的点的护理新测定法的发展与广泛的医疗保健，环境和农业应用中。

<html>纸是广泛使用的范围内的制剂或等级，可以官能化以调整其性能，并且可以通过毛细作用或芯吸自主地输送流体。如果纸是用疏水物质图案化（ 例如 ，光致抗蚀剂1或蜡2），流体的芯吸可以在空间上的纸层内的控制。例如，所施加的含水样品可以定向成许多不同的区，以与存储在纸内的化学和生化试剂反应。这些纸基微流体装置已被证明是用于便携式和廉价的分析测定3，4，5，6，7的发展有用的平台。纸基微流体装置的应用包括点的护理诊断EF"&gt; 8，环境污染物9的监测，检测假药10，和离域医疗（或"远程医疗"）的有限资源设置11。 构图纸的多个层可被组装成一个集成装置，其中来自相邻层（ 即 ，高于或低于）亲水区连接，以形成连续的流体网络，其入口和出口可联接或左独立。 12每一层可以包括一个唯一的图案，使试剂和多个测定的空间分离在单个设备上执行。所得三维微流体装置，不仅能够芯吸流体，以使分析测定的（ 例如 ，肝功能试验13和小分子14的电化学检测），但它也可以SUP端口一些复杂的功能（ 例如 ，阀门15和简单机械16）常见的传统微的方法。重要的是，因为纸灯芯通过毛细作用流体，这些设备可以从用户最小的努力工作。 因为试剂可以存储在纸基装置的三维结构中，复杂的协议可以减少到单一的加入含水样品到设备。最近，我们介绍了可用于使用蜡印技术来创建图案化层纸质免疫发展的一般三维器件结构。 17，18，这些研究主要集中在与层叠的层的设备号的设计方面是如何使用的，组合物的层，并且该三维微流体网络控制的总体％的图案免疫测定的性能。最终，我们能够利用这些设计规则，以促进一个多路复用免疫测定19的快速发展。在此手稿，人类绒毛膜促性腺激素（hCG;怀孕激素）一个先前开发的免疫测定17被用作一个例子来说明，我们已对三维纸基免疫测定的装配和制造开发的策略。因此，我们专注于一个设备，而不是化验开发的组装和操作。 在夹心免疫测定，这是一种用于检测hCG的，特定于激素的一个亚基涂覆到固体基质中，然后被阻止，以限制一个样品或任何后续的试剂的非特异性吸附捕获抗体的格式。该基板是最经常的聚苯乙烯微孔板（ 例如 ，酶联免疫吸附测定或ELISA）。然后将样品加入到孔中，温育一段时间。严谨洗涤后，特定的hCG的另一亚基的抗体并使其孵育。该检测抗体可以产生可测量的信号缀合的胶体粒子，酶，或荧光团。该井再次解释的测定的结果（ 例如 ，使用读板器）之前洗涤。而商业试剂盒依靠这种耗时多步过程中，所有这些步骤都可以迅速地在纸基微流体装置以最少的干预来的用户执行。 用于hCG的免疫测定的装置，包括六个有源层，其是，从顶部到底部，用于样品此外，共轭存储，孵化，捕获，洗涤和印迹（ 图1）。试样添加层从定性滤纸制成。它有利于引入液体样品的和保护试剂在缀合物Laye的ř从由用户环境或意外接触污染。缀合物层（定性滤纸）持有用于免疫测定的色剂（ 例如 ，胶体金标记的抗体）。孵化层（定性滤纸）允许样品到纸平面内横向行进到达下一个层，捕获层之前促进与试剂的分析物的结合。所述捕获层（尼龙膜）含有特异性吸附到材料被分析物的配体。在测定完成后，该层显露以使完成的免疫复合体的可视化。洗涤层（定性滤纸）绘制过量液体，包括自由共轭试剂从脸部的捕获层的入印迹层（厚色谱纸）的距离。六层设备是由图案化的，双面粘合剂的五层保持在一起:永久性粘合剂的四层保持ASSEM的完整性放血装置以及可移除的粘合剂的一层便于剥离装置的检查捕获层上的免疫测定的结果。 对于本手稿的目的，我们使用的hCG仅阴性和阳性对照样品（0 MIU / mL和81 MIU /毫升，分别），以提供一纸基免疫分析，其允许之间的关系的一个专门讨论的代表性结果制造方法和设备的性能。除了演示如何制造设备的成功，我们强调的几个制造误差，可能导致设备或不可重复测定结果的失败。在这个手稿详细的协议，并讨论将提供有价值的洞察纸质免疫是如何设计和制造的研究人员。虽然我们集中我们的免疫证明，我们预计这里提出的指导方针将是三地扪制造广泛有用简称AE，下同纸基微流体装置。

<table><tbody><tr><td>Illustrator CC</td><td>Adobe</td><td></td><td>to design patterns for layers of paper and adhesive</td></tr><tr><td>Xerox ColorQube 8580 printer</td><td>Amazon</td><td>B00R92C9DI</td><td>to print wax patterns onto layers of paper and Nylon</td></tr><tr><td>Isotemp General Purpose Heating and Drying Oven</td><td>Fisher Scientific</td><td>15-103-0509</td><td>to melt wax into paper</td></tr><tr><td>Artograph LightTracer</td><td>Amazon</td><td>B000KNHRH6</td><td>to assist with alignment of layers</td></tr><tr><td>Apache AL13P laminator</td><td>Amazon</td><td>B00AXHSZU2</td><td>to laminate layers together</td></tr><tr><td>Graphtec CE6000 Cutting Plotter</td><td>Graphtec America</td><td>CE6000-40</td><td>to pattern adhesive films</td></tr><tr><td>Swingline paper cutter</td><td>Amazon</td><td>B0006VNY4C</td><td>to cut paper or devices</td></tr><tr><td>Epson Perfection V500 photo scanner</td><td>Amazon</td><td>B000VG4AY0</td><td>to scan images of readout layer</td></tr><tr><td>economy plier-action hole punch</td><td>McMaster-Carr</td><td>3488A9</td><td>to remove alignment holes&amp;nbsp;</td></tr><tr><td>Whatman chromatogrpahy paper, Grade 4</td><td>Sigma Aldrich</td><td>WHA1004917</td><td></td></tr><tr><td>Fisherbrand chromatography paper (thick)&amp;nbsp;</td><td>Fisher Scientific</td><td>05-714-4</td><td>to function as blot layer</td></tr><tr><td>Immunodyne ABC (0.45 &amp;micro;m pore size )</td><td>Pall Corporation</td><td>NBCHI3R</td><td>to function as material for capture layer</td></tr><tr><td>removable/permanent adhesive-double faced liner</td><td>FLEXcon</td><td>DF021621</td><td>to facilitate peeling</td></tr><tr><td>permanent adhesive-double faced liner</td><td>FLEXcon</td><td>DF051521</td><td></td></tr><tr><td>wax liner</td><td>FLEXcon</td><td>FLEXMARK 80 D/F PFW LINER</td><td>to assist with patterning adhesive</td></tr><tr><td>acrylic sheet</td><td>McMaster-Carr</td><td>8560K266&amp;nbsp;</td><td>to fabricate frame</td></tr><tr><td>self-adhesive sheets</td><td>Fellowes</td><td>CRC52215</td><td>to use as protective slip</td></tr><tr><td>absolute ethanol</td><td>VWR</td><td>89125-172</td><td>to sanitize work area</td></tr><tr><td>bovine serum albumin</td><td>AMRESCO</td><td>0332</td><td></td></tr><tr><td>Sekisui Diagnostics OSOM hCG Urine Controls</td><td>Fisher Scientific</td><td>22-071-066</td><td>to use as positive and negative samples</td></tr><tr><td>anti-&amp;beta;-hCG monoclonal antibody colloidal gold conjugate (clone 1)</td><td>Arista Biologicals&amp;nbsp;</td><td>CGBCG-0701</td><td>to treat conjugate layer</td></tr><tr><td>goat anti-&amp;alpha;-hCG antibody</td><td>Arista Biologicals&amp;nbsp;</td><td>ABACG-0500</td><td>to treat capture layer</td></tr><tr><td>10X phosphate buffered saline</td><td>Fisher Scientific</td><td>BP3991</td><td></td></tr><tr><td>Oxoid skim milk powder</td><td>Thermo Scientific</td><td>OXLP0031B</td><td></td></tr><tr><td>Tween 20</td><td>AMRESCO</td><td>M147</td><td></td></tr></tbody></table>

fabrication of three-dimensional paper-based microfluidic devices for immunoassays

纸中自主灯芯流体由于毛细管作用。通过用疏水性障碍图案的纸张，流体输送可以控制和一层纸内定向。此外，堆叠构图纸的多层创建复杂的三维微流体网络可以支持的分析和生物分析测定法的发展。纸基微流体装置是便宜的，携带方便，易于使用，并且不需要外部设备进行操作。其结果是，他们拥有巨大潜力，作为点的护理诊断的平台。为了正确评价的效用和纸为基础的设备的分析性能，必须开发合适的方法，以确保它们的制造是可重复的，并在一个尺度适合于实验室环境。在这个手稿中，一种方法来制造，可以用于纸基免疫测定一般器件结构进行说明。我们使用添加剂manufacturin的一种形式克（多层层压）来制备构成图案的纸张和图案化粘合剂的多层器件。除了演示正确使用这些三维纸基微流体装置与人绒毛膜促性腺激素（hCG）的免疫测定，在制造过程中的错误可能导致设备故障进行了讨论。我们预计这种方法制造的纸张为基础的设备会发现专门针对资源有限的设置旨在分析应用的广泛的实用性。

在三维纸基微流体装置获得可再现的测定性能依赖于确保设备之间的一致性的制造方法。为了实现这一目标，我们已经确定了一些制造工艺和材料方面的考虑，并在展示纸质免疫的情况下在这里他们讨论。我们使用的蜡印刷方法，以形成纸基微流体装置（ 图2A）内的疏水性的障碍。 2此方法是理想的，因为它仅依赖于广泛使用的办公设备，需要最少的程序步骤来完成的，并且不要求使用可能与蛋白质吸附干扰或改变纸纤维的润湿性的化学物质（ 例如 ，光致抗蚀剂）的。此外，蜡印生产的流体通路与可重复的尺寸，这是至关重要的可重复的表演和持续时间TI试验MES。形成疏水性障碍后，粘合剂片材施加到层，以促进三维设备（ 图2B）的组装。用于免疫测定所需要的任何试剂可粘合膜被加入到层（ 图2C）的背面之后施加。因为许多装置可以并行制备该程序是用于在学术实验室制造工艺是有用的。之后的器件的所有层被堆叠并层压在一起（ 图2D）完成用于免疫测定装置的组装过程。我们加样开始检测。在这个例子中，我们用一个尿控制为妊娠试验设置，其中载有缓冲的hCG阴性和阳性样品，作为样品展示我们设备的操作和使用它们进行测定的再现性。洗涤缓冲液的两等份，然后依次加入。一旦洗涤缓冲液的最终等分试样已经完全进入该装置中，试验被认为是完整的。然后三甲层剥离以显示捕获层（ 图3A）。这个步骤不可逆地损害装置确保它不能被再次使用。的基于纸张的免疫结果定性颜色显示，可以显示在目视检查阴性或阳性输出完成。这些结果的客观性是在使用平板扫描仪（ 图3B）获得的未校正图像显而易见。 失败的实验可以经常突出某些程序步骤的重要性时实验的分析集中于成功的结果可能是，否则难以察觉。我们证明导致在免疫测定的故障三维纸基微流体装置的制造和组装三个错误:（ⅰ）偶尔，设备故障是看不出来的，直到化验完成之后。例如，一英里包括孵育信道和捕获区层之间salignment可引起不规则图案在正信号，这可能导致在定性信号的由用户（ 图4A）的误解的发展。 （ⅱ）如果该蜡没有以足够的量的印刷或不允许通过纸的整个厚度完全融化，然后将所得的疏水屏障的完整性可能受到影响。不完全形成的这些障碍会造成过芯吸失控，并导致在装置内泄漏。例如，代替限制流向一个层内的信道，半透蜡屏障将允许流体在纸平面别处灯芯。没有定义的信道，所述样品是不可能达到的捕获或洗涤层。用户将察觉到这类型的错误作为一个大大缩短试验持续时间。我们运用的红色食用色素的解决方案，以一拉演示这个设备故障揭掉的蜡模不准融化了整整30秒（ 图4B）。使用这样的层的免疫测定"已完成"，在6分钟，这比在15分钟的预期持续时间明显不同。 （iii）该需要的时间比预计完成可以指示在一个装置的制造故障测定。例如，不正确地切割的粘合剂或闭塞的孔由于试剂的过量（ 例如 ，封闭剂或胶体金）的应用程序可以从输入装置（ 图4C）禁止一个样品或洗涤缓冲液。 总体而言，我们的生产协议是有用的制造，其规模是一个学术实验室用平行众多纸基微流体装置。我们证明通过进行70测定在平行使用该方法制得的纸基的hCG免疫测定的性能:35负重复和35正řeplicates。出于演示的目的，我们准备了一套层用我们的设备的设计，贴纸层用粘合剂，然后切薄片到设备中的行。各片材切​​成7行，其中载有十台设备。这促进了层的排列上，其中这些层被录音，然后与来执行分析所需要的试剂处理的较小的丙烯酸类帧。装置制备的此方法建议在协议的说明。以下各层的治疗中，这些设备被组装在十条，然后层压。最终器件的制造步骤完成后，该装置仍然在十条和样品加入到每个设备。我们观察到对于设备0％的失败率使用我们的协议制造。我们使用的开放源码的图像处理软件20来量化这些分析的结果。虽然有许多方法可用来分析的强度distribut离子在圆形斑点（ 例如 ，放射状或直线分布）21，我们测量来自使用整个检测点作为感兴趣的区域中的装置的一个RGB图像的绿色通道的平均强度。 17，18，19 然后，我们通过减去原始负数据（ 图3B）正常化的积极和消极测定的测量值。我们确定了每个数据设定为1％的变异系数为测定采用负样品进行，并使用阳性样品进行用于测定3％。 <img alt="图1" src="/files/ftp_upload/55287/55287fig1.jpg" /> 图1:图示三维纸基装置。此图显示了定义在装置内流体通路的疏水性和亲水性区域，以及PATT持有层一起永久和可移动的胶粘剂erned层。每一层是由它执行在测定的功能标记。每个图层上的红色，蓝色或绿色的轮廓表明用来制造该特定层的材料（红色:色谱纸，蓝:尼龙膜，绿色环保:厚色谱纸）。尺寸给出了在毫米装置内的每个区域。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55287/55287fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55287/55287fig2.jpg" /> 图2:用于从三维纸基微流体装置制造免疫测定过程。 （A）的正面和使用蜡印加热前后图案色谱纸的片材的背面的图像。 （B）色谱纸上支持具有图案化粘合剂的膜。施加到图案化的尼龙膜的条带的亲水区（C）的处理。使用光框和定位孔作为引导一个多层器件的条带的（D）的装配。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55287/55287fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55287/55287fig3.jpg" /> 图3:解读纸质免疫的效果。 （A）中的纸基装置的顶部三个层向后剥离，以暴露捕获层和解释了测定的结果。 （B）绒毛膜促性腺激素纸质免疫性能的图形表示。描绘的结果是70次重复的平均值同时进行，其中35复制每个被用于FOř的hCG的阳性和阴性样品。误差棒代表数据组的标准偏差。未修正的，有代表性的图像从一个hCG的免疫描绘阳性（红色）和负（白色）结果示上述其各自的数据。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55287/55287fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55287/55287fig4.jpg" /> 图4:制造误差的例子。 （A）由于捕获层以上的横向通道的未对准，所述正信号被集中在读出区的一个小区域。 "湿"的圆形区域（虚线轮廓）可以观察到从与捕获层（左侧）未对准的横向通道之间的接触而产生的读出区的右侧。一个积极的读出图像正确对准的装置（右）的捕获层。 （B）在整个层的厚度蜡的不完全熔化可能导致在装置内泄漏。食用色素已被添加到该溶液中，以协助在不完全或完全形成的疏水屏障层样品的可视化。 （C）的不正确切割粘合剂可以阻止纸张，其中停止样品的流动层之间的流体网络。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55287/55287fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55287/55287fig5.jpg" /> 图5:制造三维纸基微流体装置。示意图示出了装配并构图纸的多层层压成完成三维器件。在这个例子中，70元件c一个可同时进行。的粘合剂和对准孔层已经从原理图，为了简化的目的移除。组装之后，单个设备可以被删除，在实验中使用。每个图层上的红色，蓝色和绿色的轮廓表明用来制造该特定层的材料（红色:色谱纸，蓝:尼龙膜，绿色环保:厚色谱纸）。的比例尺=25毫米除了单独的装置（右），其中有12×28 平方毫米的尺寸。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55287/55287fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays at JoVE.com

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

我们详细介绍的方法来制造三维纸基微流体装置用于在免疫测定的开发使用。我们对设备组装方式是一种多层，添加剂制造。我们展示了一个夹心免疫提供对这些类型的纸张为基础的设备的代表性结果。

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 ...

fabrication-three-dimensional-paper-based-microfluidic-devices-for

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

15.6K 次观看.  Tufts University. 我们详细介绍的方法来制造三维纸基微流体装置用于在免疫测定的开发使用。我们对设备组装方式是一种多层，添加剂制造。我们展示了一个夹心免疫提供对这些类型的纸张为基础的设备的代表性结果。 

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

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

In this manuscript, periodate oxidation and glutaraldehyde cross-linking methods for covalent immobilization of antibodies on paper discs are presented. The binding activity of immobilized antibodies was evaluated. Based on these results, a glutaraldehyde cross-linking method was used to develop a novel paper-based immunoassay for immunoglobulin G (IgG) detection.

共价结合的抗体，以纤维素纸光盘及其裸眼比色法免疫分析应用

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) ...

科研

生物化学

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

制造的UV-Vis和拉曼光谱免疫平台

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

基于纸张的设备进行隔离外囊泡与表征

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

生物工程

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.

This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

生物医学微流体折射率匹配器件的研制

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices ...

工程学

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 ...

Design and Development of a Three-Dimensionally Printed Microscope Mask Alignment Adapter for the Fabrication of Multilayer Microfluidic Devices

This project aims to develop an easy-to-use and cost-effective platform for the fabrication of precise, multilayer microfluidic devices, which typically can only be achieved using costly equipment in a clean room setting. The key part of the platform is a three dimensionally (3D) printed microscope mask alignment adapter (MMAA) compatible with regular optical microscopes and ultraviolet (UV) light exposure systems. The overall process of creating the device has been vastly simplified because of the work done to optimize the device design. The process entails finding the proper dimensions for the equipment available in the laboratory and 3D-printing the MMAA with the optimized specifications. Experimental results show that the optimized MMAA designed and manufactured by 3D printing performs well with a common microscope and light exposure system. Using a master mold prepared by the 3D-printed MMAA, the resulting microfluidic devices with multilayered structures contain alignment errors of &lt;10 &#181;m, which is sufficient for common microchips. Although human error through transportation of the device to the UV light exposure system can cause larger fabrication errors, the minimal errors achieved in this study are attainable with practice and care. Furthermore, the MMAA can be customized to fit any microscope and UV exposure system by making changes to the modeling file in the 3D printing system. This project provides smaller laboratories with a useful research tool as it only requires the use of equipment that is typically already available to laboratories that produce and use microfluidic devices. The following detailed protocol outlines the design and 3D printing process for the MMAA. In addition, the steps for procuring a multilayer master mold using the MMAA and producing poly(dimethylsiloxane) (PDMS) microfluidic chips is also described herein.

This project allows small laboratories to develop an easy-to-use platform for the fabrication of precise multilayer microfluidic devices. The platform consists of a three-dimensionally printed microscope mask alignment adapter using which multilayer microfluidic devices with alignment errors of &lt;10 &#181;m were achieved.

设计和开发用于制造多层微流体设备的三维打印显微镜面膜对齐适配器

This project aims to develop an easy-to-use and cost-effective platform for the fabrication of precise, multilayer microfluidic devices, which typically ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

基于磁性纳米颗粒免疫分析的交错限制微流控亚克力装置的计算机数控微铣削

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

纳米纤维素纸基分析装置的微压花制备  

Microembossing: A Convenient Process for Fabricating Microchannels on Nanocellulose Paper-Based Microfluidics

Nanopaper, derived from nanofibrillated cellulose, has generated considerable interest as a promising material for microfluidic applications. Its appeal lies in a range of excellent&#160;qualities, including an exceptionally smooth surface, outstanding optical transparency, a uniform nanofiber matrix with nanoscale porosity, and customizable chemical properties. Despite the rapid growth of nanopaper-based microfluidics, the current techniques used to create microchannels on nanopaper, such as 3D printing, spray coating, or manual cutting and assembly, which are crucial for practical applications, still possess certain limitations, notably susceptibility to contamination. Furthermore, these methods are restricted to the production of millimeter-sized channels. This study introduces a straightforward process that utilizes convenient plastic micro-molds for simple microembossing operations to fabricate microchannels on nanopaper, achieving a minimum width of 200 &#181;m. The developed microchannel outperforms existing approaches, achieving a fourfold improvement, and can be fabricated within 45 min. Furthermore, fabrication parameters have been optimized, and a convenient quick-reference table is provided for application developers. The proof-of-concept for a laminar mixer, droplet generator, and functional nanopaper-based analytical devices (NanoPADs) designed for Rhodamine B sensing using surface-enhanced Raman spectroscopy was demonstrated. Notably, the NanoPADs exhibited exceptional performance with improved limits of detection. These outstanding results can be attributed to the superior optical properties of nanopaper and the recently developed accurate microembossing method, enabling the integration and fine-tuning of the NanoPADs.

作者聚焦：通过纳米纸上的微通道制造彻底改变微流体 

This protocol describes a straightforward process that utilizes convenient plastic micro-molds for simple microembossing operations to fabricate microchannels on nanofibrillated cellulose paper, achieving a minimum width of 200 &#181;m.

微压花：在纳米纤维素纸基微流控上制造微通道的便捷工艺

Nanopaper, derived from nanofibrillated cellulose, has generated considerable interest as a promising material for microfluidic applications. Its appeal ...

三维纸基微流体装置的制造用于免疫分析

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微压花：在纳米纤维素纸基微流控上制造微通道的便捷工艺