这项工作是由韩国国家研究基金会（NRF）赠款（2013R1A2A2A05004314，2012R1A1A2043747），来自韩国的健康科技研发项目，卫生部和福利（A121994）和IBS-R020-D1的授予由韩国政府资助的支持。

注:血从健康个体抽取并收集在一个血液收集管。所有志愿者签署知情同意书。 1. 二氧化钛NF垫的研制<ol><li> 的前体溶液 22 制备 <ol><li>溶解1.5克在乙醇（99.9％，3毫升）和冰醋酸（3毫升）的混合物四异丙醇钛（TTIP），并在磁力搅拌器上在RT（25℃）30分钟的溶液混合。 </li><li>溶解在3.64克的乙醇（99.9％）聚乙烯基吡咯烷酮（PVP）的11％（重量），并用热板磁力搅拌器搅拌，在65℃下该溶液1小时。 </li><li>结合并搅拌在65℃下将两种溶液，在热板磁力搅拌器4小时。 </li></ol></li><li> 静电 <ol><li>加载制备溶液成连接到200μm内diame的不锈钢针的注射器之三。除去柱塞后倾直接溶液进入注射器。 </li><li>不断推出该溶液在在高DC电压（15千伏）放置在接地的基底为0.3毫升/小时10分钟的流速到Si晶片（2毫升×2厘米）。在电纺丝中，设置在针和接地衬底到10厘米之间的距离。 </li></ol></li><li> 在NF垫煅烧 <ol><li>放置的NF垫在炉中，以3℃/分钟的高真空条件下（5×10 -5托 ​​）下一个斜坡速率增加温度高达500℃。 </li><li>保持在500℃的温度下3小时。在炉内冷却样品至室温（25℃）的温度。 </li></ol></li></ol> 2.二氧化钛的集成2 NF垫成一个微流控离心光盘<ol><li> 盘的制造 <ol><li>使用3D设计软件（ 例如 ，索尔idWorks或类似）来描绘腔室，通道，或光盘的各层的阀门。使用代码生成软件（ 例如 ，Edgecam公司或类似）转换的设计文件到计算机数控（CNC）的代码。根据说明书使用软件说明书23,24注:本设备中使用的典型的通道尺寸为1.0毫米×0.3毫米（宽x深）。 </li><li>开放式数控铣床的操作软件（ 例如 ， DeskCNC或类似）并装载数控代码，操作软件，然后点击为了如下:"AUTO" - &gt;"G代码OPEN" - &gt;选择生成数控代码。 </li><li>装上数控铣床上的PC板:选用1 mm印刷板为光盘层顶层5毫米PC板。运行数控铣床通过点击"开始"按钮剪切光盘的各层。 </li></ol></li><li> 的 TiO 2 的NF垫 转移 到盘<ol><li>放置一个硅衬底和含有40微升（十三氟-1,1,2,2-四氢辛基）-1-三氯硅烷在真空条件下的真空室30分钟小培养皿。所述氟硅烷蒸发并得到涂布在基材上。 </li><li>混合聚二甲基硅氧烷（PDMS）预聚物与10的固化剂:1的比例和脱气在一个真空室中。旋涂的PDMS到在650的转速为60秒的硅衬底。 </li><li>固化在烘箱中涂覆的硅基材上的PDMS在65℃下10分钟以实现粘附的状态。 注:固化时间可根据环境条件而改变，粘附力可通过使用流变仪一粘性试验进行测试。 </li><li>传输通过静电和煅烧制得的二氧化钛NF垫子上使用镊子涂PDMS硅。然后，手动应用的转移TiO 2的NF垫的保形压力带平面物体（ 例如 ，载玻片），并取出异物。放该样品在烘箱中在65℃下4小时或在80℃1小时以完全固化的PDMS层。 </li><li>在20微升的PDMS /固化剂混合物在盘涂层结合反应室（10:1），其充当粘合剂层对于TiO 2的NF垫附着在盘上。 </li><li>分配50微升上的PDMS层上的二氧化钛的NF垫乙醇（99.9％），切开的NF垫用6毫米直径的冲头孔，和TiO 2的NF垫传送切涂有20微升结合反应室在先前步骤中的PDMS。 注意:在此步骤中，乙醇将有助于从Si衬底分离切断的 TiO 2的NF垫。 </li><li>孵育在烘箱中的 TiO 2的NF垫集成圆盘在65℃下4小时或在80℃1小时以完全固化的PDMS。 </li></ol></li><li> 在盘上 的二氧化钛 的NF垫 的表面改性 <ol><li> P在乙醇（3-环氧丙氧基丙基）甲基二硅烷（GPDES）的repare 1％体积/体积溶液（99.9％）。 </li><li>治疗用氧等离子体在TiO 2的NF垫集成圆盘在140瓦，180秒为50sccm氧气流。分配每个纳米纤维垫100微升GPDES溶液，并在室温（25℃）孵育2小时。 </li><li>洗用洗瓶分配乙醇（99.9％）的底物短暂地，然后通过反向盘和印迹它与干净擦拭完全除去乙醇。固化在80℃下1小时。用相同的方式乙醇（99.9％）洗涤两次，如上所述，以除去物理吸附的和未结合GPDES分子。 </li><li>吹干用氮气流，在真空下干燥。 注:该光盘可以在RT（25℃）贮存在密封容器中直至使用。 </li></ol></li><li> 的表面上的抗体的固定化 <ol><li>使200微克/毫升捕获的抗体的溶液（单克隆小鼠抗人C反应蛋白或者由稀释用磷酸盐缓冲盐水（PBS）缓冲液（pH 7.4）中的抗体的单克隆小鼠抗cTnI）。免除5微升用微量中盘解决方案到每个NF垫。请参阅材料清单有关抗体的更多信息。 </li><li>保持光盘在湿润室中，并在37°C孵育4小时。洗涂的NF垫用0.1％BSA-PBS缓冲液中的抗体。填充使用微量洗涤缓冲液100微升室，由要把孔内的缓冲区。最后，反转光盘并吸干对清洁擦拭，然后组装光盘。 </li></ol></li><li> 盘总成 <ol><li>绘制使用CAD程序（ 例如 ，AutoCAD或类似）上双面胶带盘的设计。启动CAD设计到刻字机。 </li><li>切使用切割机的双面胶带。揭下双面胶带的一个保护层，并附加我T于光盘层的顶部。剥离其他保护层和盘层上附着的顶层。 </li><li>加载预先组装盘的压制机和精确地对准使用每个层对齐标记来连接每个阀，通道和腔室顶部/粘合剂/光盘层。使用压面机应用适形压力。 </li></ol></li></ol> 3.免疫<ol><li>补使用微量用1％BSA-PBS缓冲液的室中，并在37°C孵育盘1小时。通过使用微量吸液除去缓冲区。执行此步骤以阻止未反应位点，并减少在盘蛋白质的非特异性吸附。 </li><li>通过填充和使用微量吸移室用0.1％BSA-PBS洗涤两次。 注意:在这个阶段，盘可以储存在4℃直至使用。 </li><li>负载10的抗原掺入的全血或无CRP血清为在盘上的CRP检测微升使用micropipett即用于制备校准曲线，使用的CRP的浓度从1微克/毫升到100纳克/毫升;和肌钙蛋白从10微克/ ml至100ng / ml的。 注意:由于在更高的水平在全血，这通常是在μM范围的CRP，无CRP的血清被用于证明所述装置的检测下限。 </li><li>旋转盘在3600转（391 XG）60秒以分离红细胞。 </li><li>开阀＃1通过激光照射，并传送4微升上清液血浆到包含8微升的由2400转速旋转光盘3秒检测与HRP缀合的抗体的腔室。 注意:阀门驱动和盘操作的可视化的一般流程进行了详细以往报告中描述的21 </li><li>适用的混合模式（15赫兹-1，15°）为5秒的蛋白质和检测抗体的结合。 </li><li>开阀＃2，并且在步骤6中制备的混合物转移到结合反应室（2 400转，3秒）。然后，应用一个混合模式（60赫兹-1，2°）20分钟以实现所述混合物并在TiO 2的会员协会的结合的抗体之间的免疫反应。该反应中，开阀＃3后，残余的混合物转移到废物腔室（2 400转，10秒）。 </li><li>通过打开阀＃4和纺丝盘（2 400转，4秒）传送洗涤缓冲液（600微升）到结合反应室中。然后进行120秒的应用混合模式（30赫兹-1，30°）洗涤二氧化钛会员协会在腔室。 </li><li>通过以下步骤3.8的相同的条件下洗涤的 TiO 2会员协会两次，并通过2400转速旋转光盘20秒完全除去剩余的洗涤缓冲液。然后，关闭阀门＃5。 </li><li>对于化学发光反应，时间为1分钟施加一个混合模式（30赫兹-1，2°）通过打开阀＃6和纺丝的化学发光底物溶液，以结合反应室转印后盘2400转，持续10秒之后。 </li><li>通过打开阀＃7和2400的转速旋转光盘10秒转移反应的底物溶液到检测腔室。 </li><li>测量使用的光电倍增管（PMT）模块装备检测系统反应的底物溶液的化学发光信号。 注:该检测系统是使用以下部分的定制机器:步进电机，光学机械组件，翻译阶段，并且可商购的PMT。的光学机械部件和步进电机组装到保持装置，并且该电机能转动盘。该检测是通过光电倍增管进行固定在光学机械部件，它位于上述装置的检测腔室。通过操纵步进电机，检测腔室的正下方PMT的检测区对齐。 </li></ol>

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The authors have nothing to disclose.

TiO 2的NF集成光盘上的检测是存在于血液量非常低低丰度蛋白进行超灵敏检测快速，廉价和方便的技术。该技术具有使用小样品体积（10微升）的优点，并且是适合用于同时多个样品的分析。这提供了巨大的潜力作为一个复用免疫测定装置。该装置具有附加的优点，它不需要像血浆分离，这需要在常规的ELISA样品预处理步骤。此外，该设备可以执行整个免疫测定程序自动地（ 例如 ，混合，洗涤，装订等）由于预先设计的腔室，通道和阀门。此外，使用市售的光盘制造方法使用粘合剂（ 例如 ，注塑成型，紫外/超声波/热粘接）代替研磨和手工装配，从设备制造到分析整个过程可以是自动化。 此外，这里介绍的转印印刷法示出了从供体静电的NF的简单和容易传输通过利用薄粘合剂的PDMS层到目标衬底。在一般情况下，在煅烧过程之后获得的TiO 2的会员协会是脆性的，易于从接地基底剥离由于弱的附着力。25由于其特性脆性，与其它设备集成在TiO 2的NF垫是困难的。为了解决这个问题，已经报道了许多方法已经。例如，热压26和溶剂蒸气27的技术表明煅烧过程之前TiO 2的会员协会垫和目标表面之间的密合性的提高。尽管这些方法增加了粘合性，在TiO 2的NF垫并非完好由于高机械压力或各技术中施加溶剂。此外，由于在煅烧步骤的，在TiO 2的集成&lt;/ SUB&gt;使用这些方法NF垫只能在有限的目标面被应用。 据我们所知，这是上显示到由热塑性塑料的设备的NF垫转印印刷，即使在转印后保留会员协会的新颖特性的第一种技术。对于该装置的更好的性能，高品质的会员协会的制造和预固化条件的优化是期望的。在协议中所提到的，所需的预固化可根据环境条件而变化的时间;因此，预固化条件是通过使用粘性测试测量粘附力被优化。 此外，该协议这里提出，提供熟练导向为在盘上的完全自动化的ELISA方法。该协议没有介绍光盘的唯一制造，而且所有ELISA所必需的过程，包括全血分离，计量，混合，清洗和检测的自动化。每个步骤与纺丝速度，混合频率和操作时间最优化。根据本操作指南上，装在盘上的试剂可以利用单个马达来传送。由于该装置的血液非常低的体积中进行优良的检测灵敏度，它提供了通过在疾病的早期阶段筛选指标诊断应用的巨大潜力。该装置将被激活取决于其特异性抗体的可用性，以检测任何生物标志物。 虽然用的 TiO 2的NF垫的装置来实现高度增强的灵敏度，由于垫的表面积大，这种技术的剩下的挑战之一可以是在大量生产均匀的NF垫。因为在ELISA的灵敏度强烈影响纳米纤维垫的表面积，它是实现不仅高的表面积，而且在大量生产的不同批次可重现和一致的表面面积的关键。 </p&gt; 总之，我们已经表明一个简单的方法来脆性NF垫集成到超灵敏蛋白质检测功能元件。此方法的优点如下:1）预先静电纺丝的 TiO 2会员协会只能在导电和热稳定的表面来制备这里，我们已经表明，它们可以被转移到任何基质上，包括非导电塑料材料; 2） 二氧化钛的NF垫能够承受压力和期间，由于PDMS层的高粘附力的洗涤步骤保持稳定; 3）它提供了生物测定相对较高表面积和4）抗体可以共价连接到TiO 2的会员协会由于在NFS完整表面性质。该技术具有很大的潜力可用于纳米纤维材料的融入了多种应用设备。

基于纳米材料的1,2-如纳米线，3纳米颗粒，4纳米管，5和纳米纤维（NFS）6-8疾病诊断多种平台已经制定出来。这些纳米材料提供新技术的设计，由于其独特的物理化学性质高度敏感的生物测定极好的前景。例如，氧化物中孔锌纳米纤维已经被用于乳腺癌的生物标志物的毫微微摩尔灵敏的检测。9最近，基于二氧化钛（TiO 2）已探索生物分析应用10考虑到它们的化学稳定性的纳米材料，11微不足道蛋白质变性，12和生物相容性。13另外， 二氧化钛的表面上的羟基基团促进 ​​化学修饰和生物分子的共价连接。14,15图案化的 TiO 2 THIN薄膜16或TiO 2纳米管17已被用于通过增加表面积以增强靶蛋白的检测灵敏度;然而，制造工艺是相当复杂的，并且需要昂贵的设备。另一方面，静电会员协会受到关注，因为它们的高表面积以及简单和低成本的制造过程; 18,19然而，该静电的 TiO 2的NF垫的易碎或松散特性使得它难以处理和与微流体装置因此集成。6,20，在TiO 2的NF垫中的生物分析应用很少使用，特别是那些需要苛刻的洗涤条件。 在这项研究中，要克服这些限制，我们已经开发出一种新技术，用于通过利用​​一个薄聚二甲基硅氧烷（PDMS）粘合剂层的静电NF垫转印到任何目标衬底的表面上。 Furthermore，我们已经成功地显示出静电的 TiO 2的NF垫的整合到由聚碳酸酯（PC）的离心微流体装置。使用这种装置，高敏感的，完全自动化，并集成检测C-反应蛋白（CRP）以及心肌肌钙蛋白I（cTnI水平）溶液在30分钟内从仅10微升的全血21来实现的。由于该组合NFS和离心平台的性能优势，该测定显示出六个数量级的宽动态范围从1微克/毫升（〜8 FM）到100纳克/毫升（〜下午12点08分）与检测的下限0.8微克/毫升（〜6 FM）为CRP和100纳克/毫升（〜4纳米）为37微克/毫升（〜下午1点05分）的检测限的动态范围从10微克/毫升（~0.4 PM） cTnI浓度。这些检测限〜300和〜20倍下相比，他们的相应的常规ELISA结果。这种技术可应用于用于检测任何靶蛋白，用适当的抗体。总体来说，这款设备合作ULD大大有助于在体外诊断和生物化学试验，因为它可以从非常少量的生物样品甚至检测罕见量的靶蛋白具有高度的敏感性; 例如 ，10微升的全血。尽管我们仅展示在这项研究中使用ELISA血清蛋白的检测，静电会员协会与微流体设备的传输和集成技术，可以更广泛地用于需要高检测灵敏度大表面积其他生化反应的应用。

<table><tbody><tr><td>Si wafer</td><td>LG SILTRON</td><td>Polished Wafer, test grade</td><td>Dia. (mm) = 150, orientation = &amp;lt;100&gt;, dopant = boron, RES(Ohm-cm) = 1 - 30, thickness (&amp;mu;m) = 650 - 700</td></tr><tr><td>Polycarbonate (PC)&amp;nbsp;</td><td>Daedong Plastic</td><td>PCS#6900</td><td>Thickness (mm) = 1 and 5&amp;nbsp;</td></tr><tr><td>Titanium tetraisopropoxide, 98%,</td><td>Sigma-Aldrich</td><td>205273</td><td></td></tr><tr><td>Polyvinylpyrrolidone, Mw = 1,300,000</td><td>Sigma-Aldrich</td><td>437190</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>320099</td><td></td></tr><tr><td>Anhydrous ethanol</td><td>Sigma-Aldrich</td><td>459836</td><td></td></tr><tr><td>Tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane</td><td>Sigma-Aldrich</td><td>448931</td><td></td></tr><tr><td>PDMS and curing agent</td><td>Dow Corning</td><td>SYLGARD 184</td><td></td></tr><tr><td>GPDES</td><td>Gelest Inc</td><td>SIG5832.0&amp;nbsp;</td><td></td></tr><tr><td>Ethanol</td><td>J T Baker</td><td></td><td></td></tr><tr><td>FE-SEM</td><td>FEI</td><td>Nova NanoSEM</td><td></td></tr><tr><td>X-ray photoelectron spectroscopy</td><td>ThermoFisher</td><td>K-alpha</td><td></td></tr><tr><td>3D modeling machine</td><td>M&amp;amp;I CNC Lab, Korea</td><td></td><td>CNC milling machine</td></tr><tr><td>Wax-dispensing machine</td><td>Hanra Precision Eng. Co. Ltd., Korea</td><td></td><td>Customized</td></tr><tr><td>Double-sided adhesive tape</td><td>FLEXcon, USA</td><td>DFM 200 clear 150 POLY H-9 V-95</td><td></td></tr><tr><td>Cutting plotter</td><td>Graphtec Corporation, Japan</td><td>Graphtec CE3000-60 MK2</td><td></td></tr><tr><td>Spin coater</td><td>MIDAS</td><td>SPIN-3000D</td><td></td></tr><tr><td>Furnace (calcination)</td><td>R. D. WEBB COMPANY</td><td>WEBB 99</td><td></td></tr><tr><td>Rheometer (Tack test)</td><td>Thermo Scientific</td><td>Haake MARS&amp;nbsp;&lt;font face=&quot;Arial&quot; size=&quot;4&quot;&gt;III&lt;/font&gt; - ORM Package</td><td></td></tr><tr><td>Oxygen plasma system</td><td>FEMTO</td><td>CUTE</td><td></td></tr><tr><td>Monoclonal mouse antihuman hsCRP</td><td>Hytest Ltd., Finland</td><td>4C28</td><td>(clone # C5)</td></tr><tr><td>Monoclonal mouse anti-cTnI</td><td>Hytest Ltd., Finland</td><td>4T21</td><td>(clone # 19C7)</td></tr><tr><td>HRP conjugated goat polyclonal anti-hsCRP</td><td>Abcam plc., MA</td><td>ab19175</td><td></td></tr><tr><td>HRP conjugated mouse monoclonal anti-cTnI</td><td>Abcam plc., MA</td><td>ab24460</td><td>(clone # 16A11)</td></tr><tr><td>hsCRP</td><td>Abcam plc., MA</td><td>ab111647</td><td></td></tr><tr><td>cTnI</td><td>Fitzgerald, MA</td><td>30-AT43</td><td></td></tr><tr><td>Bovine Albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>PBS</td><td>Amresco Inc</td><td>E404</td><td></td></tr><tr><td>Blood collection tubes</td><td>BD vacutainer</td><td>367844</td><td>K2 EDTA 7.2 mg plus blood&lt;br/&gt; collection tubes</td></tr><tr><td>SuperSignal ELISA femto</td><td>Invitrogen</td><td>37074</td><td></td></tr><tr><td>Modular multilabel plate reader</td><td>Perkin Elmer</td><td>Envision 2104</td><td></td></tr><tr><td>Disc operating machine</td><td>Hanra Precision Eng. Co. Ltd., Korea</td><td></td><td>Customized</td></tr><tr><td>Photomultiplier tube (PMT)</td><td>Hamamatsu Photonics</td><td>H1189-210</td><td></td></tr><tr><td>AutoCAD</td><td>AutoDesk</td><td>Version 2012</td><td>Design software</td></tr><tr><td>SolidWorks 3D CAD software&amp;nbsp;</td><td>SOLIDWORKS Corp.</td><td>Version 2013</td><td>3D Design software,</td></tr><tr><td>Edgecam</td><td>Vero software</td><td>version 2009.01.06928</td><td>Code generating software</td></tr><tr><td>DeskCNC</td><td>Carken Co.</td><td>version 2.0.2.18</td><td>CNC milling machine software</td></tr></tbody></table>

fully automated centrifugal microfluidic device for ultrasensitive protein detection from whole blood

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

使用该协议，从全血中的高灵敏度制备蛋白质检测一个完全自动化的离心微流体装置。在TiO 2的NF垫由静电和煅烧的方法制备。为了制造所需直径，形态和厚度的NFS，静电如流量，电压，和纺纱的时间条件进行了优化。当条件不优化，形成在NFS的质量很差。特别是，NFS未从在低电压（5千伏）的聚合物溶液纺出，并且当电压为高被打破（&gt; 20千伏）。同样，纺纱时间对NFS的制造与合适的密度，不要过于稀疏（1分钟）或过于密集（30分钟）的关键。另外，为了避免造成高流速珠形成，流速操纵。这样，15千伏的施加电压，10分钟静电时间和0.3ml hr的流速<suP&gt; -1用作基于形态学和所得会员协会的厚度（ 图1）对NF制造的最佳条件。 使用粘合剂的PDMS层，其制备通过旋涂的PDMS硅烷化的硅基板，并在65℃下预固化它在烘箱中对TiO 2的NF垫被成功转移到目标衬底。预固化的PDMS层时通过检查粘接力，使用粘性试验（ 图2A）进行了优化。 图2B示出上纳米纤维附着固化时间的效果。如果与PDMS进行3分钟的加热，不存在粘附力作为PDMS仍然未固化和保持为液体并在NFS得到嵌入到PDMS层。当它被加热30分钟，粘合力是非常弱的PDMS变得僵硬和失去其粘性属性和在NFS未附着于它。当PDMS加热FOR 10分，PDMS表现出较强的附着力和会员协会的强烈连接。从结果来看，可以得出结论，用于固化预PDMS为强附着的最佳时间为10分钟。 圆盘组件之后，免疫测定在TiO 2的NF垫集成盘上通过如表1中所列以下一系列盘操作过程进行的。对于未知样品中的CRP和cTnI的浓度的确定，对于每个校准图表是通过标绘制成相对光单位（RLU）与所述CRP或肌钙蛋白浓度（ 图3）。在图3A和3B所示，盘上测定结果与常规的ELISA对分别CRP和cTnI的一个96孔板进行比较。测定法表现出广泛的线性动态范围的为CRP的0.8微克/毫升（〜6 FM）和37微克/毫升（1.5分）为肌钙蛋白一个盘上的检测极限，这高CRP的约300倍和cTnI的20倍相比，它们各自的传统ELISA结果（286微克/毫升，2.3分用于CRP; 824微克/毫升，32日下午进行的cTnI）。 <img alt="图1" src="/files/ftp_upload/54143/54143fig1.jpg" /> 图1.静电条件的优化。在每个条件SEM照片显示形成会员协会的形态。 <a href="https://www.jove.com/files/ftp_upload/54143/54143fig1large.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/54143/54143fig2.jpg" /> 图2.粘性粘合剂的PDMS层和被转印的 TiO 2 的NF垫 的SEM图像的测试 （A）的粘合剂的粘附力PDMS层，预固化的时间数的长度，（B）的附着在固化的时间长度不同的PDMS纳米纤维的SEM图像。这个数字已经从参考修改。 21. <a href="https://www.jove.com/files/ftp_upload/54143/54143fig2large.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/54143/54143fig3.jpg" /> 图3. CRP和cTnI水平的使用上实验室一个盘和96孔板检测校准曲线图。CRP的检测掺入在无CRP的血清（A）和肌钙蛋白全血中（B）的掺入进行的。误差棒表示至少三次独立测量的标准偏差。检出限（LOD）的限制，通过用无CRP血清或全血测定未经肌钙蛋白尖峰为阴性对照数据的标准偏差的3倍计算C反应蛋白和肌钙蛋白，分别为。 <a href="https://www.jove.com/files/ftp_upload/54143/54143fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 自旋号 </td><td> 转速（rpm） </td><td> 阀编号 </td><td> 时间（秒） </td><td colspan="4"> 手术 </td></tr><tr><td> 1 </td><td> 3600 </td><td></td><td> 60 </td><td colspan="4">血液分离</td></tr><tr><td> 2 </td><td> 2400 </td><td> 1 </td><td> 3 </td><td colspan="4">开阀到等离子体转移到含8微升的检测抗体的腔室与HRP缀合</td></tr><tr><td> 3 </td><td> 15赫兹，15° </td><td></td><td>五</td><td colspan="4">混合血浆和检测抗体</td></tr><tr><td> 4 &lt;/ TD&gt; <td> 2400 </td><td> 2 </td><td> 3 </td><td colspan="4">开阀的混合物转移到结合反应室</td></tr><tr><td>五</td><td> 60赫兹，2° </td><td></td><td> 1200 </td><td colspan="4">在TiO 2 NF垫和等离子检测抗体的混合物混合捕获抗体</td></tr><tr><td> 6 </td><td> 2400 </td><td> 3 </td><td> 10 </td><td colspan="4">打开阀以除去混合物</td></tr><tr><td> 7 </td><td> 2400 </td><td> 4 </td><td> 4 </td><td colspan="4">开阀转让洗涤液</td></tr><tr><td> 8 </td><td> 30赫兹，30° </td><td></td><td> 120 </td><td colspan="4">混合洗涤液洗TiO 2的NF垫</td></tr><tr><td> 9 </td><td> 2400 </td><td></td><td> 4 </td><td colspan="4">转让洗涤液</td></tr><tr><td> 10 </td><td> 30赫兹，30° </td><td></td><td> 120 </td><td col跨度="4"&gt;混合洗涤液洗TiO 2的NF垫</td></tr><tr><td> 11 </td><td> 2400 </td><td></td><td> 4 </td><td colspan="4">转让洗涤液</td></tr><tr><td> 12 </td><td> 30赫兹，30° </td><td></td><td> 120 </td><td colspan="4">混合洗涤液洗TiO 2的NF垫</td></tr><tr><td> 13 </td><td> 2400 </td><td></td><td> 4 </td><td colspan="4">转让洗涤液</td></tr><tr><td> 14 </td><td> 30赫兹，30° </td><td></td><td> 120 </td><td colspan="4">混合洗涤液洗TiO 2的NF垫</td></tr><tr><td> 15 </td><td> 2400 </td><td></td><td> 20 </td><td colspan="4">除去剩余的洗涤缓冲液</td></tr><tr><td> 16 </td><td></td><td>五</td><td></td><td colspan="4">关闭阀门</td></tr><tr><td> 17 </td><td> 2400 </td><td> 6 </td><td> 10 </td><td colspan="4">开vALVE并传送化学发光底物</td></tr><tr><td> 18 </td><td> 30赫兹，2° </td><td></td><td> 60 </td><td colspan="4">混合基板上的TiO 2 NF垫的免疫试剂</td></tr><tr><td> 19 </td><td> 2400 </td><td> 7 </td><td> 10 </td><td colspan="4">打开阀和反应衬底转移到检测室</td></tr><tr><td colspan="3">总时间</td><td> 〜30分钟</td><td colspan="4"></td></tr></tbody></table> 表1.一种用于在光盘的免疫操作程序。该表已经从参考修改。 21。

Watch this Scientific Journal Video about Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood at JoVE.com

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

全自动离心微流体装置从全血超灵敏蛋白检测

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

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

fully-automated-centrifugal-microfluidic-device-for-ultrasensitive

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.4K 次观看.  School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), UNIST-gil 50, Ulsan, Republic of Korea. This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 µL of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

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

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

基于毛细管的离心微流体装置单分散微滴的大小可控的形成

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

科研

工程学

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 ...

生物工程

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

微流体平台精密小批量来样加工及它的使用尺寸分离生物颗粒的声微型

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

数字微流体进行自动蛋白质组学处理

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

生物学

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and endothelial cells, provide an ideal platform for an updated status on the immune condition of an individual. The microfluidic protocol allows for enrichment of CNCs without selective cell loss and sample preparation variability due to user-mediated steps. Briefly, the protocol includes device fabrication, sample collection, device setup, and running blood through the microfluidic chamber. Within the device whole blood is rapidly mixed with deionized water for approximately 10 seconds in a 50 micron x 150 micron microfluidic channel. In this time span erythrocytes are lysed due to hypotonic conditions. Herringbone structures on the bottom of the channel ensure thorough mixing and exposure of cells to a constant environment. Remaining cells are returned to isotonic conditions at the exit of the device, fixed using 2% paraformaldehyde, centrifuged to separate erythrocyte debris from CNCs, and suspended in flow buffer for staining and analysis by flow cytometry. Results show clean flow cytometry scatter plots with CNC populations saved. Significance of this device and protocol comes in the study and understanding of disease pathogenesis by analysis of CNC populations. Hence, automation, effectiveness, and simplicity of the microfluidic protocol are demonstrated.

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

微流控自动化血液循环核细胞富集的裂解协议

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

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 ...

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 ...

A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection

Source: Sage, J. M., et al. <a href="https://app.jove.com/v/60638">Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates a capillary electrophoresis immunoassay protocol for detecting a protein biomarker associated with amyotrophic lateral sclerosis (ALS) from human blood platelets. Confirmation of the presence of the ALS biomarker protein is achieved through size-based separation of the platelet proteins followed by chemiluminescent detection.

用于蛋白质生物标志物检测的毛细管电泳免疫测定

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of buffers and ...