作者承认罗伯特·W·德基金会，国防威胁降低局（DTRA）和美国国家科学基金会新兴前沿研究和创新（EFRI）的资金支持。作者还感谢马里兰州Nanocenter及其Fablab洁净室设施的支持。

1，微细加工的微流控芯片的<ol><li>准备电化学芯片<ol><li>图案金电极<ol><li>冲洗空白4&#39;硅晶片（黄金级品质）用丙酮，甲醇，异丙醇（"急性心肌梗死"干净）。冲洗从去离子水（DI），随后用N 2枪干燥该晶片的异丙醇。 </li><li>生长出1μm厚的SiO 2钝化层用等离子增强化学气相沉积（PECVD）的工具。存200了厚厚的一层铬其次是2000了厚厚的一层黄金直流溅射工具。 </li><li>自旋"PR1"正光致抗蚀剂（纺丝参数:3000转，1000转/秒，30秒），以创建均匀的膜〜1.6微米厚。预烘烤的晶片在100℃下1分钟的热板上。 </li><li>揭露了190兆焦耳/ 平方厘米，在通过光掩模405纳米波长的晶片。开发晶圆30秒352开发eloper和冲洗晶圆与DI和干与N 2枪。 </li><li>蚀刻金腐蚀剂金1.5分钟。蚀刻铬蚀刻剂〜30秒的铬。冲洗用去离子晶圆干燥与N 2枪。 </li><li>剥去了"急性心肌梗死"清洁过程中的光致抗蚀剂。 注:强氧化剂及有爆炸危险。 </li><li> 1 H 2 SO 4:1分钟的 H 2 O 2的混合物（"食人鱼"干净）与4清洗晶片。冲洗用去离子晶圆干燥与N 2枪。 </li></ol></li><li>图案铂电极<ol><li>自旋"PR2"正光致抗蚀剂（纺丝参数:3000转，1000转/秒，30秒），以创建均匀的膜〜1.4微米厚。预烘烤的晶片在100℃下1分钟的热板上。 </li><li>揭露了30兆焦耳/ 平方厘米，在通过光掩模405纳米波长的晶片。烘烤晶片45秒，在125℃下的热板上。洪水暴露该晶片拥有1000兆焦耳/ 平方厘米在405 nm波长处无光罩。开发晶片在6 2分:1 AZ400K开发商和冲洗用去离子晶圆干燥与N 2枪。 </li><li>沉积400埃厚的钛层，接着用电子束蒸发系统1600厚厚的一层铂。 </li><li>抬离光致抗蚀剂中的超声波处理丙酮浴中5分钟，然后冲洗，用丙酮和异丙醇。冲洗用去离子晶圆干燥与N 2枪。 </li></ol></li></ol></li><li>准备含量频道<ol><li>准备模具的含量频道<ol><li>冲洗空白4&#39;硅晶片（测试等级质量）与"急性心肌梗死"清洁程序。冲洗从用DI晶片的异丙醇，然后用N 2枪干燥。 </li><li>自旋"PR3"负光致抗蚀剂（2步，纺丝参数:步骤1 - 600转，120转/秒，10秒步骤2 - 1150转，383转/秒，27秒），以创建均匀的膜〜100μm厚。预烘烤的热板上（2步烘烤参数晶片:步骤1 - 从室温上升到65℃，在300℃/小时，并保持温度10分钟，第2步 - 斜坡上升至95℃在300℃/小时，并保持温度30分钟）。 </li><li>揭露2500兆焦耳/ 平方厘米，在通过光掩模405纳米波长的晶片。后烘焙晶片逐渐升高至95℃，以300℃/小时，并保持温度有关的热板上10分钟。开发的晶片为在丙二醇单甲醚乙酸酯（PGMEA）显影10分钟。漂洗，用异丙醇和干燥用N 2枪晶片。 </li></ol></li><li>伪造检测通道<ol><li>制备10:1聚二甲基硅氧烷（PDMS）混合物（20克弹性体，2克固化剂）和完全脱气的真空室20分钟。 </li><li>限定的模晶片周围的外壳用铝箔和慢慢倾未固化的PDMS在模具中。 </li><li>烘烤的PDMS在箱式炉中模具（2步烘烤参数:步骤1 - 5分钟，从室温上升到80℃，第2步 - 保持在80℃下为17分钟）。 </li><li>剥掉PDMS从铝箔模具和地点。切开法芯片的外科医生刀，定义孔活检冲压工具。 </li></ol></li></ol></li></ol> 2，装配设备<ol><li>手动对齐PDMS测定通道的电化学芯片上的工作电极。 PDMS枝以及电化学芯片，密封该通道，并防止泄漏。 </li><li>柔性管（外径0.087&#39;及身份证0.015&#39;）连接到一个塑料弯管或直连接器使用的是短挠性管作为适配器（外径0.1875&#39;及身份证0.0625&#39;）。附加连接器连接到每个设备中的穿有孔的入口。 </li><li>连接注射器上的另一端油管，并将注射器注射泵。根据所要求的程序步骤在第3和其相应的溶液中交替地改变设定注射器，管，和一个连接器。 </li></ol> 3，分析DNA杂交请注意:在介绍每个溶液慢慢进入通道以200微升/小时的流速，直到整个信道被充满。 <ol><li>功能化具有不同的单链DNA探针序列每个垂直微通道（并行执行的3个独立的垂直微）: <ol><li>填充每个微通道具有不同的单链DNA探针保温溶液（10mM磷酸盐缓冲液，100 mM氯化钠，10μM磷酸三（2 - 羧乙基）膦（TCEP），和1μM探针的单链DNA），并孵育1小时，然后漂洗所述微通道用PBS。 </li><li>填充微通道用含6 - 巯基-1 - 己醇（MCH）1mM的在10mM的PBS中，100mM的NaCl和1.395毫TCEP的缓冲液中的溶液，并保温（护）软化为1小时，随后漂洗，用PBS将微通道。 </li></ol></li><li>抬离PDMS，旋转90°，水平方向，并将其放置下来，露出了反应室分开行包含一个独特的电极和参比电极（图S1）每一行。 </li><li>填具有4倍的控制测量溶液中的微通道浓缩柠檬酸盐钠（SSC）缓冲液，并温育20分钟。 </li><li>背景测量:填充的微通道用5mM铁氰化钾/ 5mM的亚铁氰化物的PBS溶液，并记录该电化学系统的不同频率的阻抗值（电化学阻抗谱; EIS）使用连接到工作，计数器恒电位，以及参考电极（ EIS参数:频率范围为1 MHz至0.1赫兹，每十年10个频率点的数据，25 mV的幅度，5 mV的极化与参比电极）。重复此测量中的微通道的每个工作电极。 </LI&gt; <li>测量DNA杂交（对于每个非互补的，互补的单链DNA靶执行）。 <ol><li>填充微通道具有4倍的目标单链DNA测定溶液浓缩含有1μM的目标单链DNA的SSC缓冲液，并温育20分钟。测量DNA根据步骤3.4。 </li></ol></li></ol>

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Acta. 643, 26-37 (2009).</li><li>Arinaga, K., Rant, U., Tornow, M., Fujita, S., Abstreiter, G., Yokoyama, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+surface+charging+during+the+coadsorption+of+mercaptohexanol+to+DNA+layers+on+gold:+Direct+observation+of+desorption+and+layer+reorientation.">The role of surface charging during the coadsorption of mercaptohexanol to DNA layers on gold: Direct observation of desorption and layer reorientation.</a> Langmuir. 22, 5560-5562 (2006).</li><li>Katz, E., Willner, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+biomolecular+interactions+at+conductive+and+semiconductive+surfaces+by+impedance+spectroscopy:+Routes+to+impedimetric+immunosensors,+DNA-sensors,+and+enzyme+biosensors.">Probing biomolecular interactions at conductive and semiconductive surfaces by impedance spectroscopy: Routes to impedimetric immunosensors, DNA-sensors, and enzyme biosensors.</a> Electroanalysis. 15, 913-947 (2003).</li></ol>

作者什么都没有透露。

我们的方法展示出一种微流体为基础的电化学生物芯片的制造和利用的DNA杂交的事件分析。通过高收益率来控制微细加工过程中，我们开发包括集成电化学传感器阵列微型通道的设备。我们已经通过迭代的方法设计用于电化学芯片和微流体通道模具的光刻过程控制处理参数。这些步骤为其他微型分析仪器未来创造的指导方针。重要的是，它们提供了一种方法，以优化各种生物感测的参数，如信号 ...

微流控芯片实验室级芯片（LOC）的设备提供众多优势，在临床诊断，环境监测和生物医学研究。这些器件利用微流体通道，以控制流体流动的芯片，其中各种程序可发生包括试剂的混合，基于结合亲和力，信号转导和细胞培养1-4的区域。微流控技术提供了许多优点比传统的临床诊断工具，如微孔板读者或电泳凝胶迁移实验。微流体器件需要的大小为2〜3个数量级（纳升，而不是微升）更少的试剂来执行类似分析。此外，这些设备可以增加通过其中的一些生物学事件的发生是由于在物种内的通道5,6越小约束的速度。第三，传感器可以在使用光刻技术和蚀刻技术的微流体装置，其可以提供无标记detectio被集成ñ。最后，这些装置是廉价的，以产生与需要的操作7-10技术人员的部分少的工作。 标签的检测通常是使用光学或电气传感器执行。光学器件可以是由于与样品中的分析物低干扰呈现更好的感测性能。然而，他们的表现受到损害的情况下样品的背景具有相同的共振波长的传感器11。有许多优点，使用电信号来执行生物和化学检测在微流体系统中。制造本质上是较不复杂的，因为这些传感器通常只需要图案化电极来操作。此外，电信号可以直接与具有最高的测量设备，而其它信号模式可能要求换能器的信号12-15转换。电子传感器通常测量的变化impedancê16,17，电容18，或氧化还原活性19。然而，新的挑战表示为这些系统的小型化。要克服的最重要的挑战包括:样品制备和流体（由于低的样品体积和雷诺数）的混合，物理和化学作用（包括毛细管力，表面粗糙度，建筑材料和分析物之间的化学相互作用），低信号 - 噪比20-23，并从复杂的生物样品（ 例如 ，血液和唾液）中电活性分析物的潜在干扰（通过减小的表面积和体积。生产）。这些影响进一步的调查将导致在可重复的方式处理这些设备，将改善其整体性能的精确制造和操作指南。 用于DNA杂交检测广泛诊断遗传性疾病24,25和各种形式的CANC呃26。每年，流感多株被确定使用从DNA杂交技术，结果27例患者。流感病毒就占每年36,000人死亡，在美国28。这样的例子可能受益于在不牺牲灵敏度或特异性可以执行相同的分析技术如用低的样品体积的板读取器或凝胶移位分析，并在成本的一小部分台式微流体装置。由于无标记的电化学传感的许多优点，它已被广泛地用于检测DNA杂交事件29,30。其中宏观尺度的电极（在毫米范围内）浸在烧杯中与感兴趣的溶液A的设置可以被用于提供关于单链DNA序列的结合动力学它们匹配的互补序列非常敏感数据。最近，一直在结合电化学传感MICR几进展ofluidics的DNA杂交。研究已经完成对杂交动力学31和传感器整合在微流体通道检测15。然而，仍然需要一种能够分析DNA杂交事件在平行无需复杂的样品制备步骤的快速高通量微流体装置。 在这项工作中提出的设备提供了一个平台，允许多个交互并行，没有复杂的样品制备步骤进行筛选。我们的协议介绍如何微流体为基础的电化学生物芯片的微细加工和微机电系统（MEMS）技术32,33。我们描述的两个微流体芯片，聚二甲基硅氧烷制成（PDMS），以及电化学芯片的制造过程中，由电极阵列。生物芯片的化学官能与单链DNA探针也被解决。最后，ABIL生物传感器的性特异性检测和分析的ssDNA目标是证明。总体而言，微流体为基础的电化学生物芯片是一种快速和高通量分析技术。它可以被用于研究生物分子和导电换能器之间的相互作用，并能在各种上实验室芯片应用中使用。

<table><tbody><tr><td>4&#039;&#039; Silicon wafer</td><td>Ultrasil</td><td>4-5664</td><td>Single side polished; P-type; Boron doped; Orientation 1-0-0; Thickness 500 micron; Resistance 10-20 ohm&amp;middot;cm</td></tr><tr><td>Shipley 1813 photoresist (&quot;PR1&quot;)</td><td>Microchem</td><td></td><td>positive photoresist</td></tr><tr><td>AZ5214 photoresist (&quot;PR2&quot;)</td><td>Hoechst Celanse</td><td></td><td>positive photoresist</td></tr><tr><td>SU-8 50 photoresist (&quot;PR3&quot;)</td><td>Microchem</td><td></td><td>negative photoresist</td></tr><tr><td>Gold etchant</td><td>Transene</td><td>TFA</td><td>No dilution</td></tr><tr><td>Chromium etchant</td><td>Transene</td><td>1020AC</td><td>No dilution</td></tr><tr><td>PDMS elastomer</td><td>Dow Corning</td><td>3097366 1004</td><td></td></tr><tr><td>PDMS curing agent</td><td>Dow Corning</td><td>3097358 1004</td><td></td></tr><tr><td>Biopsy punching tool</td><td>Healthlink</td><td>BP20</td><td></td></tr><tr><td>Tygon flexible tubing</td><td>Cole Parmer&amp;nbsp;</td><td>R 3603</td><td>0.015&quot;</td></tr><tr><td>Tygon flexible adapter</td><td>Cole Parmer&amp;nbsp;</td><td>06417-41</td><td>0.0625&quot;</td></tr><tr><td>1 ml syringe</td><td>Beckton-Dickenson</td><td>301025</td><td></td></tr><tr><td>Monobasic potassium phosphate</td><td>Fluka</td><td>1551139</td><td></td></tr><tr><td>Potassium phosphate dibasic anhydrous</td><td>Sigma</td><td>RES20765-A7</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>tris(2-carboxyethyl)phosphine</td><td>Aldrich</td><td>C4706</td><td></td></tr><tr><td>6-mercapto-1-hexanol</td><td>Aldrich</td><td>451088</td><td></td></tr><tr><td>20x concentrated saline-sodium citrate buffer</td><td>Sigma</td><td>93017</td><td></td></tr><tr><td>Potassium hexacyanoferrate(III)</td><td>Aldrich</td><td>455946</td><td></td></tr><tr><td>Complementary ssDNA #1</td><td>Integrated DNA Technologies (IDT)</td><td>100 nmole DNA oligo</td><td>5&amp;rsquo;-GGGACGTCCACGGAGCGCTA&lt;br/&gt; TCGGAGCTTT-3&amp;rsquo;</td></tr><tr><td>Target ssDNA #2</td><td>Integrated DNA Technologies (IDT)</td><td>100 nmole DNA oligo</td><td>5&amp;rsquo;-/5ThioMC6-D/ACGCGTCAGGTCATTGACGA&lt;br/&gt; ATCGATGAGT-3&amp;rsquo;</td></tr><tr><td>Complementary ssDNA #2</td><td>Integrated DNA Technologies (IDT)</td><td>100 nmole DNA oligo</td><td>5&amp;rsquo;-ACTCATCGATTCGTCAATGA&lt;br/&gt; CCTGACCCGT-3&amp;rsquo;</td></tr><tr><td>Target ssDNA #3</td><td>Integrated DNA Technologies (IDT)</td><td>100 nmole DNA oligo</td><td>5&amp;rsquo;-/5ThioMC6-D/ACCTAGATCCAGTAGTTAGA&lt;br/&gt; CCCATGATGA-3&amp;rsquo;</td></tr><tr><td>Complementary ssDNA #3</td><td>Integrated DNA Technologies (IDT)</td><td>100 nmole DNA oligo</td><td>5&amp;rsquo;-TCATCATGGGTCTAACTACT&lt;br/&gt; GGATCTAGGT-3&amp;rsquo;</td></tr><tr><td colspan="4">&lt;strong&gt;Instrument&lt;/strong&gt;</td></tr><tr><td>Plasma Enhanced Chemical Vapor Deposition (PECVD)</td><td>Oxford Instruments</td><td>PlasmaLab System 100</td><td>Chamber pressure 1,000 mTorr, Tempreture 200 &amp;#176;C, RF power 20 W, Gasses: a) N&lt;sub&gt;2&lt;/sub&gt;O flow rate 710 sccm; b) a composition of 5% SiH&lt;sub&gt;4&lt;/sub&gt; and 95% N&lt;sub&gt;2&lt;/sub&gt; gasses flow rate 170 sccm, SiO&lt;sub&gt;2&lt;/sub&gt; growth rate 690 &amp;#197;/min.</td></tr><tr><td>DC sputtering unit</td><td>AJA International</td><td>ATC 1800-V</td><td>For Chrome: Chamber pressure 10 mTorr, Argon flow rate 20 sccm, Supplied DC power 200 W, Sputter rate 10 nm/min. For Gold: Chamber pressure 10 mTorr, Argon flow rate 20 sccm, Supplied DC power 200 W, Sputter rate 36 nm/min.</td></tr><tr><td>E-beam evaporation system</td><td>Denton</td><td>Custom-built</td><td>For Titanium: Chamber pressure &amp;lt; 2 x 10&lt;sup&gt;-6&lt;/sup&gt; Torr, Supplied power to the e-gun 7.5 kW, Filament current 150-200 mA, Evaporation rate 6-10 &amp;#197;/sec. For Platinum: Chamber pressure &amp;lt; 2 x 10&lt;sup&gt;-6&lt;/sup&gt; Torr, Supplied power to the e-gun 7.5 kW, Filament current 150-200 mA, Evaporation rate 2-3 &amp;#197;/sec.</td></tr><tr><td>Potentiostat</td><td>CH Instruments</td><td>660D</td><td></td></tr><tr><td>Syringe pump</td><td>KD Scientific</td><td>KDS230</td><td></td></tr></tbody></table>

a microfluidic-based electrochemical biochip for label-free dna hybridization analysis

分析台式程序进入微规模的小型化提供显著优点在关于反应时间，成本和集成的预处理步骤。利用这些设备对的DNA杂交事件的分析是重要的，因为它提供了一种技术，生物标志物的实时评估，在点的护理各种疾病。然而，当该装置的足迹减少的各种物理现象的增加的优势地位。这些现象影响了制造精度和设备的运行可靠性。因此，存在很大的需求，以改善总体性能精确制造和操作中的可重现的方式处理这些装置。在这里，我们描述的协议和用于制造和微流体为基础的电化学生物芯片的DNA的杂交事件的准确分析的操作方法。生物芯片是由两部分组成:一个微流体芯片与三个平行的微通道进行聚二甲基硅氧烷（PDMS），以及一个3×3阵列的电化学微芯片。采用电化学阻抗谱（EIS）分析，检测到的DNA杂交的事件。 EIS的分析，可以在电化学系统中是主要的，在这些尺度的特性的监视的变化。与以监测电荷转移和扩散阻力与生物传感器的变化的能力，我们证明以下20分钟的选择性互补的单链DNA靶，380纳米的计算检测限，并且13％的交叉反应性与其他非互补单链DNA孵化。这种方法可以通过阐明上扩散在微观尺度制度的行为，以及允许的DNA杂交事件的研究提高微型设备的性能。

可控和精确的制造工艺进行实验装置的研究是必不可少的。这使研究人员能够获得重复性和高通量实验。在这里，我们表现出了高产量，高重复性的微细加工过程中的微流体为基础的电化学生物芯片（ 图1）。具有故障率低，一些设备已经表明，导致溶液的泄漏结合的问题。为了验证生物芯片的电化学活性，循环伏安法测量在充有电活性的氧化还原对铁氰化物/亚铁氰化物的微通道进?...

Watch this Scientific Journal Video about A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis at JoVE.com

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

我们提出了一个微流体为基础的电化学生物芯片的DNA杂交检测。以下的ssDNA探针官能化，特异性，灵敏度和检测极限进行了研究具有互补和非互补ssDNA的目标。结果表明在电化学系统中的DNA杂交事件的影响，以3.8纳米的检测极限。

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

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

a-microfluidic-based-electrochemical-biochip-for-label-free-dna

Research

JoVE Journal

Bioengineering

17.0K 次观看.  University of Maryland. 我们提出了一个微流体为基础的电化学生物芯片的DNA杂交检测。以下的ssDNA探针官能化，特异性，灵敏度和检测极限进行了研究具有互补和非互补ssDNA的目标。结果表明在电化学系统中的DNA杂交事件的影响，以3.8纳米的检测极限。 

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

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

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 ...

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 ...

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

生物学

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

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 ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

基于电润的数字微流体平台，用于自动酶链接免疫吸附测定

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Microfluidic Capillary Electrophoresis for Fragment Sizing of PCR Products: A Microchip-Based Electrophoretic Separation Technique to Separate DNA Fragments Based on Size

Source: Wang, H., Zhu et al. <a href="https://www.jove.com/v/59963">A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene</a>. J. Vis. Exp. (2019).
This video describes the microfluidic capillary electrophoresis technique to separate PCR amplicons based on their size. This technique helps in fragment sizing using fluorescent dyes and standard molecular size markers.

用于 PCR 产物片段大小的微流体毛细管电泳:一种基于微芯片的电泳分离技术，可根据大小分离 DNA 片段

Source: Wang, H., Zhu et al. A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Automated Capillary Electrophoresis Based Fragment Size Distribution Analysis of cfDNA: A DNA Microchip-based Method to Study Size Distribution of DNA Fragments Isolated from Biofluids

Source: Palande, V. et al. <a href="https://www.jove.com/video/61449" target="_blank">Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients.</a> J. Vis. Exp. (2020)
In this video, we analyze the size distribution of cell-free DNA or cfDNA fragments using automated capillary electrophoresis. This technique helps gain insights into the profile of DNA fragments released by cancer cells during apoptosis.

基于自动毛细管电泳的 cfDNA 片段大小分布分析:一种基于 DNA 微芯片的方法，用于研究从生物流体中分离的 DNA 片段的大小分布

Source: Palande, V. et al. Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients. J. Vis. Exp. (2020)
In this video, we analyze the size ...

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

摘要

探索更多视频

此视频中的章节

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

微流控芯片的制备及方法检测甲型流感

一个微流控芯片单细胞的多功能化学分析

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装

与复用电子检测的粒子的空间跟踪微流体平台

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

基于电润的数字微流体平台，用于自动酶链接免疫吸附测定

数字核酸定量实验分析的简单堆积读数

用于 PCR 产物片段大小的微流体毛细管电泳:一种基于微芯片的电泳分离技术，可根据大小分离 DNA 片段

基于自动毛细管电泳的 cfDNA 片段大小分布分析:一种基于 DNA 微芯片的方法，用于研究从生物流体中分离的 DNA 片段的大小分布

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

摘要

探索更多视频

此视频中的章节

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

微流控芯片的制备及方法检测甲型流感

一个微流控芯片单细胞的多功能化学分析

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

与复用电子检测的粒子的空间跟踪微流体平台

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

基于电润的数字微流体平台，用于自动酶链接免疫吸附测定

数字核酸定量实验分析的简单堆积读数

用于 PCR 产物片段大小的微流体毛细管电泳:一种基于微芯片的电泳分离技术，可根据大小分离 DNA 片段

基于自动毛细管电泳的 cfDNA 片段大小分布分析:一种基于 DNA 微芯片的方法，用于研究从生物流体中分离的 DNA 片段的大小分布

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装