这项研究得到了美国国立卫生研究院（NIH）的资助R01 EB008268。

1。芯片制造12 <ol><li>车型两个斑块从ZEONEX 690R粒料：8-9克ZEONEX粒料均匀地分配在中心上的金属板，预热5分钟，在198℃下加热的压榨，然后慢慢地施加压力2500 psi的另外5分钟。要完成这一步，我们使用了卡弗热压。 </li><li>浮雕微流体通道中的牌匾用环氧模。在模具上制造的详情概述别处12（在图2b中的通道设计）：把一个斑块上的环氧模;预热他们的热压机上，在157℃下进行10分钟，然后慢慢地施加压力至1,000 psi另一个10分钟的。从模具用手工或用钳子冷却下来之前删除的牌匾。 （ 注意：在此步骤中，戴上隔热手套）。 </li><li>钻孔，在入口处，废弃口，和微流体通道的出口的压花芯片。我们使用的1.25毫米直径ETER钻头的三个孔。 </li><li>债券压花芯片与另一斑块（顶部）：洗净芯片用IPA，RNA酶客和去离子水的顺序;空气干燥和预热它们在131℃的热压机上10分钟，并然后按在350 psi的另一个10分钟。 </li><li>附加NanoPorts是在入口处，浪费，而且出油口，分别用JB焊接环氧治愈15至24小时，每制造商的说明。 </li><li>冲洗SPE通道与50μLRNA酶客场，然后用100μl无核酸酶的水。 </li><li>装入SPE 4微升的的接枝溶液（3％w / v的二苯甲酮与甲基丙烯酸甲酯）的信道，然后通过在UV烘箱中在365nm的紫外线的波长和2000毫焦/厘米2下孵育10分钟，交联的甲基丙烯酸酯。与真空除去残余的接枝溶液。我们使用的壁真空。为了获得最佳结果，应使用新鲜溶液。它通常是可以接受的，然而，存储制备的溶液中，在4℃达一周。 </li><li>使SPE柱溶液（所有的v / v％的）：16％的乙二醇二甲基丙烯酸酯，24％甲基丙烯酸丁酯，42％的1 - 十二烷醇，18％环己醇中，并添加1％，2 - 二甲基氨基-4 - （甲基 - 苯基氨基） - 苯酚作为光引发剂。 </li><li>取出硅胶微球溶液相同体积的溶液（作出的1:1混合物）作为固相萃取柱，并完全干燥，在70℃下在真空烘箱中过夜。分手的沉淀攻管盖上盖子。添加到干燥的二氧化硅微球管和旋涡混合固相萃取柱的解决方案。 </li><li> 4微升的SPE溶液装入到相同的信道，然后交联，通过UV照射2.5分钟。然后，翻转芯片和照射为2.5分钟。聚合固相萃取柱应该是白色不透明的固体。 </li><li>洗净用500μl的100％甲醇中洗去过量的反应物的通道。洗也消除内聚合物宝rogenic溶剂，从而创造了开放的孔隙结构在环境条件下干燥时的固相萃取柱。 </li><li>将两个薄膜加热器的芯片与导热胶带的底部。加热器的两侧，需要对齐的变性和退火分开（参见图1）的边缘的宽的通道。 </li><li>止地点5成的芯片通过死的热电偶的每个芯片的开口侧通道（参见图2b）。留在这些渠道，通过压合的热电偶。磁带被用于进一步固定的位置的热电偶。 </li></ol> 2。固相萃取法<ol><li>准备96μl的70％乙醇，用DEPC处理过的和高压灭菌的水（去离子或Millipore过滤）。 </li><li>加入4微升25X核糖核酸安全的96μl的70％乙醇，在一个管中，并重复相同的混合物用100％乙醇在一个单独的管。 </li><li>准备300微升通过混合下列：75微升6M的硫氰酸胍（GuSCN），150μl的2 - 丙醇，63微升的无核酸酶的水，和12微升25X核糖核酸安全通道缓冲器。 </li><li>准备312微升的裂解缓冲液通过混合：100微升6 M GuSCN，200μl的2 - 丙醇，和12μl25X核糖核酸安全。 </li><li>热的70％乙醇，100％乙醇中，信道缓冲液和裂解缓冲液，在60℃下10-20分钟激活RNA安全。 </li><li>要平衡的微流体通道，在0.8毫升/小时的流速通过SPE通道运行通道缓冲区。 </li><li>快速解冻在VTM测试样品在37℃水浴中，并立刻从水浴中取出样品，只要它被解冻。 </li><li>离心样品在13,000 rpm离心10分钟，并转移入300μl的裂解缓冲液中加入100μl的上清液。完成该混合物通过加入6微升1微克/微升的载体RNA。 </li><li>涡混合，分割后为5秒，加载整个裂解成鲁尔乐1毫升注射器。 </li><li>运行溶解物通过SPE通道在流速为0.8毫升/小时。 </li><li>洗净SPE信道，然后用100μl的70％乙醇100微升of100％的乙醇中，在1毫升/小时。 （50μL死体积的注射器针头，不会延后，所以实际上只有50微升通过的通道）。 </li><li>定位在0.5毫升标记一个空的注射器，并推动空气通过该信道，以干燥在1毫升/小时。 </li><li>运行67.5微升的无核酸酶的水，通过该信道，以洗脱结合的核酸，以0.5毫升/小时，并收集13.5微升从nanoport在废物端口。 （流体损失是由于注射器尖端和SPE信道的54微升的死体积。） </li></ol> 3。 RT-PCR方法<ol><li>准备36.5微升的RT-PCR主混合物在空气中干燥工序（2.12）中的Qiagen公司的一步法RT-PCR试剂盒中使用的试剂。 </li><li>加载RT-PC在废弃口的nanoport，ŕ试剂和混合RNA得到以下50μLRT-PCR反应：13.5微升洗脱RNA在无核酸酶的水，4μLRT-PCR酶混合物，10微升Q的解决方案，10μl1X一步骤缓冲液，2μl的25mM的MgCl 2，1μl的50μM的正向引物5&#39;-GAC CRA TCC TGT CAC CTC TGA C-3&#39;，1微升50μM的反向引物5&#39;-AGG GCA TTY TGG ACA AAK CGT CTA-3 &#39;，2微升的dNTP，0.75微升1.0％w / v的BSA，0.73微升PEG8000和5.02微升无核酸酶的水。 </li><li> RT-PCR的混合物装入到的RT信道通过温和真空（我们使用的壁真空）和密封的Nanoport与一个封闭的拟合。 </li><li> 35 V加热器1。 RT中的信道保持试剂加热器1在50℃下平衡30分钟后保持的试剂中的RT信道为15分钟后，在95℃下的加热器1达到平衡</li><li> 27 V加热器1和50 V加热器2，等待约3分钟或直到加热器1达到平衡，在60和德克，C和加热器2的平衡，在95℃下</li><li>推到PCR试剂0.5μl/ min的通道。这需要大约20分钟，试剂流经蛇形通道。 </li><li>作为样品，使得它的蛇形通道的端部，收集的PCR产物的出口处使用一个适当大小的移液器和小费。 </li></ol> 4。 PCR产品检测<ol><li>我们使用的是安捷伦高灵敏度DNA测试，以检测产品。为了运行这个测试的安捷伦高灵敏度DNA试剂盒从冰箱中30分钟测试前。 </li><li>生物分析仪打开并打开2100专家软件。 </li><li>装入1微升的PCR产物进入测试和遵循安捷伦的协议（ <a href="http://gcf.pbrc.edu/docs/Agilent/Agilent%20Manual.pdf" target="_blank">http://gcf.pbrc.edu/docs/Agilent/Agilent％20Manual.pdf</a> ）。 </li><li>分析，并记录数据。 </li></ol>

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作者声明没有竞争的金融利益。

这里给出的诊断方法表现出的能力的集成微流控塑料芯片从患者标本放大A型流感RNA，具有较高的特异性和检出限低， 我们设计该芯片潜在照顾测试点：（一）温度和流体控制被简化，（b）该芯片是成本低，适合于高吞吐量的制造，使用注射成型，及（c）的芯片是一次性的，并且预期的一次性使用，从而降低样品的交叉污染的关注。 虽然我们目前的芯片上的运行时?...

据报道，在20世纪的三次流感大流行的数以百万计的人死亡。此外，最近的流感大流行是在2009年由世界卫生组织（WHO）宣布，截至2010年8月1日报道了世界卫生组织3，18,449人死亡。再次证明了这一流行病的高负担的传染性疾病，需要进行流感快速，准确的检测，实现快速疾病的确认，适当的公共卫生反应和有效的治疗方法4。 广泛用于诊断流感的方法有好几种，包括快速免疫测定，直接荧光抗原测试（DFA）和病毒培养方法。快速免疫显着缺乏敏感性5-8，而其他两种方法是劳动密集和耗时9。分子检测提供了多种优势，包括短匝左右的时间，高SENSITivity，和较高的特异性。的一些商业机构已着手对快速分子检测（也称为核酸检测或NAT）的传染性疾病，和一些有流感检测管线。但他们中的大多数需要关闭芯片的样品制备。没有放弃的分子测试将样品制备到试验盒或模块的临床实验室改进修正案（CLIA）。 实验室上的一个芯片技术点医疗检测设备的发展起着重要的作用。出台后的第一个PCR芯片在1993年10，无数的努力，已投入开发核酸检测芯片。然而，只有少数的这些集成的粗样品制备与下游扩增。 之前我们已经证实成塑料微流体芯片11的小型化的固相萃取柱（SPE）和开发和优化的连续流PCR芯片12。在这里，我们扩展了以前的工作整合到一个芯片上，为临床诊断和SPE与RT和PCR步骤展示其能力，从病人鼻咽部拭子和吸出物（NP）核酸扩增。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 1 - 十二烷醇</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 443816-500G </td></tr><tr><td> 2,2 - 二甲氧基-2 - 苯基苯乙酮</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 196118-50G </td></tr><tr><td> 2100生物分析仪</td><td>安捷伦科技公司，加利福尼亚州圣克拉拉</td><td> G2943CA </td></tr><tr><td> 2 - 丙醇</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 19516 </td></tr><tr><td>二苯甲酮</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 239852-50G </td></tr><tr><td> BSA </td><td>赛默飞世尔科技，pittsburge，PA </td><td> A7979-50ML </td></tr><tr><td>甲基丙烯酸丁酯</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 235865-100毫升</td></tr><tr><td>运营商RNA </TD&gt; <td> Qiagen公司，瓦伦西亚，CA </td><td> 1017647 </td></tr><tr><td>环己醇</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 105899-1L </td></tr><tr><td>乙醇</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> E7023 </td></tr><tr><td>乙二醇二甲基丙烯酸酯</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 335861 </td></tr><tr><td>乙二醇二甲基丙烯酸酯</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 335681-100ML </td></tr><tr><td>玻璃注射器250μL </td><td>汉密尔顿，内华达州里诺市</td><td> 81127 </td></tr><tr><td>异硫氰酸胍</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 50981 </td></tr><tr><td>高灵敏度DNA试剂盒</td><td>安捷伦科技公司，加利福尼亚州圣克拉拉</td><td> 5067-4626 </td></tr><tr><td>热压机</td><td>卡弗，旅游Wabash，IN </td><td> 4386 </td></tr><tr><td> JB焊接环氧树脂</td><td>的麦克马斯特-CaRR，艾姆赫斯特，IL </td><td> 7605A11 </td></tr><tr><td>露儿乐注射器</td><td>美国新泽西州的富兰克林湖，BD医疗， </td><td> 309628 </td></tr><tr><td>氯化镁</td><td>赛默飞世尔科技，pittsburge，PA </td><td> AB-0359 </td></tr><tr><td>甲醇</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 494437 </td></tr><tr><td>甲基丙烯酸甲酯</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> M55909 </td></tr><tr><td> Nanoport </td><td>厄普丘奇科学</td><td> N-333-01 </td></tr><tr><td> Nanoport配件</td><td>厄普丘奇科学</td><td> F-120X </td></tr><tr><td>无核酸酶的水</td><td>赛默飞世尔科技，pittsburge，PA </td><td> PR-P1193 </td></tr><tr><td>一步法RT-PCR试剂盒</td><td> Qiagen公司，瓦伦西亚，CA </td><td> 210210 </td></tr><tr><td> PEG8000 </td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 41009 </td></tr&gt; <tr><td>电源</td><td> VWR，宾夕法尼亚州拉德诺</td><td> 300V </td></tr><tr><td> RNA酶客场</td><td> Sigma-Aldrich公司，圣路易斯，密苏里州</td><td> 83931-250ML </td></tr><tr><td> RNASecure </td><td>美国应用生物系统公司，福斯特城，加利福尼亚</td><td> AM7005 </td></tr><tr><td>硅胶微球</td><td> Polysciences的，灵顿，PA </td><td> 24324-15 </td></tr><tr><td>注射泵</td><td>马萨诸塞州Holliston，哈佛仪器， </td><td> HA2000P/10 </td></tr><tr><td>导热胶带</td><td>麦克马斯特卡尔，艾姆赫斯特，IL </td><td> 6838A11 </td></tr><tr><td>热电偶</td><td>欧米茄工程学院，斯坦福，CT </td><td> 5SRTC-TT-J-40-36 </td></tr><tr><td>薄膜加热器</td><td>明科，明尼阿波利斯，明尼苏达州</td><td> HK5166R529L12A </td></tr><tr><td>紫外线交联剂</td><td> UPV，旱稻，CA </td><td>的CL-1000 </td></tr><tr><td> ZEONEX </td> &lt;TD&gt; Zeon Chemicals公司，路易斯维尔，肯塔基</td><td> 690R </td></tr></tbody></table>

microfluidic chip fabrication and method to detect influenza

快速和有效的诊断中发挥了重要作用，实现有效的病人管理和治疗，在控制传染病。在这里，我们提供了一个完整的微热塑性芯片的能力放大A型流感病毒在患者鼻咽部拭子和吸出物（NP）。当加载的患者样本，微流器件依次进行的片上的细胞裂解，RNA的纯化和浓缩步骤的固相萃取（SPE），反转录（RT）和聚合酶链反应（PCR），在RT-PCR室内，分别。终点检测使用片外生物分析仪（安捷伦科技，圣克拉拉，CA）。用于外围设备，我们使用一个单一的注射器泵进行驱动的试剂和样本，而两个薄膜加热器用作热源的RT和PCR室。该芯片被设计成单一的层和适合高吞吐量制造减少fabricati的时间和成本。微流控芯片分析各种病毒和细菌，只限于需要发现新的病原体的试剂设计提供了一个平台。

<html>一个典型的结果显示在图3为A型流感感染的鼻咽冲洗标本。由于流感病毒的不同量的每个患者标本中，PCR产物的终浓度会有所不同。一个很好的结果应具有低噪声，两个明显的阶梯峰值（35和10380 bp的）和一个单一的产物峰，在所设计的产品的尺寸（107 bp）的阳性样品。虽然产物峰，理论上应该不存在为阴性对照，我们确实观察到虚假的PCR峰在一些样品中引物二聚体的形成附近的?...

Watch this Scientific Journal Video about Microfluidic Chip Fabrication and Method to Detect Influenza at JoVE.com

Microfluidic Chip Fabrication and Method to Detect Influenza

一个集成的微流体热塑性芯片已被开发用于作为一种分子诊断。该芯片进行核酸提取，逆转录和PCR。用于制造和运行芯片的方法中描述。

微流控芯片的制备及方法检测甲型流感

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

microfluidic-chip-fabrication-and-method-to-detect-influenza

Research

JoVE Journal

Bioengineering

14.8K 次观看.  Boston University. 一个集成的微流体热塑性芯片已被开发用于作为一种分子诊断。该芯片进行核酸提取，逆转录和PCR。用于制造和运行芯片的方法中描述。

视频: Microfluidic Chip Fabrication and Method to Detect Influenza

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

科研

生物工程

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

微流体芯片ICPMS进样

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

影像引导下，血管源性微流控网络的基于激光的研制

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

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 ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...