This work was supported by National Science Foundation Award No. ECCS 1610995. The authors would like to thank the Institute of Electronics and Nanotechnology and the Parker H. Petit Institute for Bioengineering and Bioscience staff for their support in using shared facilities. The authors also would like to thank Chia-Heng Chu for his help in preparing the manuscript.

1.编码电极的设计 注意:图1a示出了微图案化电极的3-D结构。 <ol><li>设计一组四个的7位Gold码进行编码的微流体通道23。 <ol><li>构造两个线性反馈移位寄存器（的LFSR），每个代表本原多项式。 </li><li>使用的LFSR生成一个优选的一对7-m位-sequences。 </li><li>循环移位首选对米 -sequences并加入他们MOD 2，产生四种不同的Gold码。 </li></ol></li><li>设计编码电极（ 图1b）的布局。 <ol><li>将三个电极端子，在三个角表示正，负电极和参考电极。 </li><li>路线正极和负极迹线在每个微流体通道的相对两侧。 </li><li>延长的正极和负极进微流体通道作为电极指，以下的唯一分配金码（ 图1c）。 </li><li>放置参考电极中的正，负电极指之间。 </li><li>放置正极和负极痕迹远离最外层的参考电极指，以最小化的编码区外部的电传导。 </li></ol></li></ol> 2.表面电极的微细 注意:图2b示出表面电极的制造过程。 <ol><li>清洗在食人鱼溶液4英寸的硼硅玻璃晶片（98％硫酸:30％过氧化氢= 5:1）在120℃下进行20分钟，以除去所有的有机污染物。然后放置在一个200℃热板上的晶片20分钟以去除残留的水。 </li><li>晶圆转移到一个微调。分配2毫升负光致抗蚀剂到晶片并以3的速度旋转该晶片，000转40秒，以均匀涂布用1.5微米的光致抗蚀剂层的晶片。 </li><li>放置在一个150℃热板上的晶片和烘烤纺丝光刻胶1分钟。 </li><li>通过使用掩模对准一个铬掩模暴露光致抗蚀剂，以365纳米的UV光（225毫焦耳/厘米2）。 </li><li>放置在100℃的热板上的晶片和烘烤曝光的光刻胶1分钟。 </li><li>通过浸渍在光致抗蚀剂显影剂（RD6）晶片15秒开发光致抗蚀剂。轻轻喷洒去离子（DI）水和洗晶片。通过吹压缩氮气干燥。 </li><li>放置具有图案化光致抗蚀剂的晶片到一个电子束金属蒸发器，并在3×10 -6乇的与碱压力沉积20纳米厚的铬膜，接着是80纳米厚的金膜到晶片1埃/秒的沉积速率。 </li><li>浸入涂覆有金属的晶片在丙酮超声波浴设定在40千赫与100％的幅度的频率在室温回火30分钟ATURE蚀刻下面的光刻胶并完成剥离工艺。 </li><li>切块的晶片成使用常规的切割锯小块。 </li></ol> 3. SU-8模具微流体通道的制作 注意:图2a示出了模具的微流体通道的制造过程。 <ol><li>清洗和烘烤用2.1描述的相同的顺序的4英寸硅晶片。 </li><li>晶圆转移到一个微调。倾4mL的光致抗蚀剂到晶片上。涂上光刻胶的晶片。 <ol><li>旋转晶片以500rpm进行15秒。 </li><li>旋转晶片以1000rpm进行15秒。 </li><li>旋转晶片以3000rpm 60秒，以获得一个均匀地涂敷15微米厚的光致抗蚀剂层。 </li></ol></li><li>放置晶片面朝上擦拭浸泡在丙酮洁净室，并从背面和晶片的边缘去除残留光致抗蚀剂。 </li><li>传送晶片到热复吃软烘烤。首先，烘烤晶片在65℃下1分钟。然后迅速将晶片移动到95℃的热板上烘烤2分钟。 </li><li>通过一个铬掩膜通过使用掩模对准器暴露光致抗蚀剂，以365纳米的UV光（180毫焦耳/厘米2）。 </li><li>烘烤在65℃下将晶片暴露后1分钟，然后在95℃下2分钟。 </li><li>浸入开发商晶圆并轻轻摇动容器3分钟。然后，冲洗用异丙醇醇（IPA）的晶片，并通过吹压缩氮气干燥。如果一个白色的残余物出现在晶片上，再浸入入显影剂和开发用于更长的时间和干燥。 </li><li>烤上的200℃热板上的晶片30分钟完全干燥它。 </li><li>使用轮廓在整个晶片的不同位置，以确保均匀性测量图案化的光致抗蚀剂的厚度。 </li><li>通过利用汽相淀积的技术Silanize模具晶片。加入TR 200微升ichlorosilane在与8小时的SU-8模具晶片沿真空干燥器培养皿和地点。 </li></ol> 4.微流体代码设备的组装<ol><li>放置4英寸硅晶片，在一个150毫米直径的培养皿的模具中，并通过从它的边缘胶带固定。 </li><li>混合聚二甲基硅氧烷（PDMS）预聚合物和交联剂以10:1的比例，并倾将50g混合物放入培养皿。放置在真空干燥器培养皿脱气该混合物1小时，然后在65℃下固化它的烘箱中至少4小时（ 图2a）。 </li><li>用手术刀切出固化硅橡胶层，使用镊子把它剥开模具晶圆。验证的原理的设备的尺寸是约20mm×7毫米。然后通过与PDMS用于使用活检穿孔机微流体通道的入口和出口的直径为1.5毫米打孔。 </li><li>通过将我清洁PDMS部分的图案的一面T于无尘室胶带。 </li><li>通过用丙酮，IPA的去离子水漂洗清洗与表面电极的玻璃基板和使用压缩氮气干燥。 </li><li>与每个部分在设定为100毫瓦的RF等离子体发生器朝上的微机械加工的侧激活的PDMS在氧等离子体的表面和玻璃基板30秒。 </li><li>对齐用在玻璃基板上表面的电极的光学显微镜，然后将在物理接触的两个等离子体激活表面的PDMS微流体通道。 </li><li>烤上的70℃热板上的设备5分钟，朝向热板玻璃侧。 </li><li>通过焊接连接的电线电极的接触垫。 </li></ol> 5.制备模拟生物样品<ol><li>培养HeyA8人卵巢癌细胞中的RPMI 1640补充有10％胎牛血清（FBS）和1％青霉素-链霉素在5％CO 2气氛中于37℃直到他们达到80％汇合。 </li><li>使用玻璃吸管培养瓶中吸出介质。分配，然后吸出1×磷酸盐缓冲盐水（PBS）洗涤细胞。 </li><li>孵育细胞在2mL 0.05％（重量/体积）在37℃下2分钟的胰蛋白酶溶液中止贴壁细胞。然后，加入4毫升培养基中和胰蛋白酶。 </li><li>离心机在100×g下5分钟将细胞悬浮液以沉淀细胞在试管中。然后，吸上清彻底。 </li><li>轻轻吹打上下以机械解离的细胞团块重悬在1-2毫升1×PBS中的细胞。 </li><li>绘制细胞悬液少量到吸管和计数用血球细胞的数目。 </li><li>稀释用PBS将细胞悬液以10 5 -10 6个细胞/ mL的最终细胞浓度制备样品。 </li></ol> 6.运行微流控代码设备 注:网络古尔图3示出的实验装置。 <ol><li>放置的微流体码装置上的光学显微镜的阶段。 </li><li>申请使用电子函数发生器400 kHz正弦波到芯片上的参比电极。 </li><li>正和负感测电极连接到两个独立的跨阻抗放大器的电流信号从每个转换为电压信号。 </li><li>减去以获得一个双极信号使用差分电压放大器的负感测电极的电压信号的正感测电极的电压信号。 </li><li>使用高速摄像机来进行验证和表征的目的设备的光学记录操作。 </li><li>驱动通过微流体码装置中的细胞悬浮液在使用注射泵以恒定流速（50-1000微升/小时）。 </li><li>测量使用锁定放大器的阻抗调制信号。 <ol><li>参考交流信号连接到裁判该锁定放大器的erence输入。差分的双极信号连接到锁定放大器作为输入信号。 </li><li>获得来自锁相放大器输出的差分信号的RMS幅值。 </li></ol></li><li>样品在1 MHz速率的锁定放大器的输出信号转换成通过一个数据采集板用于进一步分析的计算机。 </li></ol> 7.处理传感器信号的<ol><li>传输记录电气数据到MATLAB进行后期处理和解码。 </li><li>过滤使用巴特沃斯滤波器（MATLAB内置函数），以去除高频噪声（&gt; 2.5千赫）数字域记录的信号。 </li><li>生成传感器信号模板的代码库。 <ol><li>识别对应于在该装置的每个传感器代表非重叠码信号和从数据集作为单独的波形矢量提取这些信号块。 </li><li>每个标准化模板代码波形矢量通过它的力量。使用MATLAB内置函数（bandpower）来测量信号功率。 </li><li>使用MATLAB函数（重采样）由具有变化的持续时间，以适应在细胞流速在电极变型数字创建标准化代码信号的版本以展开模板库。 </li></ol></li><li>识别对应于传感器的活动中的信号块:在滤波后的波形（阈SNR&gt; 12dB）的波形与下阈值的SNR将被视为噪声。 </li><li>通过使用基于连续干扰消除的迭代算法，在多用户CDMA通信网24，25通常采用的技术，解码中所记录的信号的传感器活性各个块。 <ol><li>计算与所有的使用滑动点积库中的模板的每个信号块的互相关。 </li><li>确定产生LARG模板EST自相关峰值以确定主个人传感器码信号。记录的自相关峰的时间和幅度。 </li><li>通过缩放基于所测量的自相关峰值幅度所确定的代码模板和定时信息（在步骤7.5.2确定）构造一个估计的传感器代码信号。 </li><li>减去从原始数据所估计的传感器代码信号。 </li><li>从7.5.1迭代的过程中，直到剩余信号没有像在模板库中的任何信号，数学上定义为相关系数小于0.5。 </li></ol></li><li>缩小步骤7.5使用一个优化过程的初始传感器信号估计。 <ol><li>通过从每次迭代增加估计个体传感器信号重建该信号。 </li><li>扫围绕原始估计各个传感器信号的幅度，持续时间和定时，以产生与记录的电信号的最佳拟合基于最小二乘逼近26。 </li></ol></li><li>通过对光学图像校准电信号转换估计传感器信号的振幅成细胞大小。 </li></ol>

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The authors have nothing to disclose.

多个电阻脉冲传感器先前已经掺入微流控芯片28，29，30，31，32。在这些系统中，电阻脉冲传感器要么不多路复用28，29，30，31或它们需要在不同的频率32被驱动单个传感器。在这两种情况下，需要有专用的外部连接的芯片上的每个电阻脉冲传感器，因此大量的传感器不能没有更大的硬件的复杂集成。微流体码的重要的优点在于，它允许从一个简单的装置中的单个输出的多个电阻脉冲传感器的同时读取。我们利用米实现这一目标电信常用ultiplexing技术来设计集成到微流体装置式微电阻脉冲传感器。从本质上说，我们的技术通过设计每个检测到的粒子时，以产生一个可区别的信号依赖于代码复用的片库尔特计数器的网络。网络中的每个微机械传感器由在不同的配置，使得与这些电极中流动的颗粒的顺序相互作用产生正交阻抗调制波形有序的多个共面的表面的电极。以适应异步粒子传感器相互作用，我们特别每个传感器，以产生Gold码33，即通常在CDMA电信网络的上行链路中使用伪正交数字码而设计的。 Gold码甚至保持一定水平的正交当它们与随机相位差34对齐。 弥crofluidic码易于扩展。虽然我们在本文中提出结果从原型微流控代码设备有四个传感器，更多的传感器可以在设备设计生产从其余部分区分开输出信号时纳入。扩大传感器网络的方法之一是基于在较大的正交码集具有更长的数字码来设计的传感器。再与更多的比特的正交码提供在解码高处理增益和可以区分彼此时，有干扰。另一方面，在该装置不再Gold码还意味着更大的感测体积，这增加了干扰传感器的预期数目。同样地，增加传感器的数目为给定样品的密度将导致更多的粒子，由于重叠的增加的总感测量。这样，样品中的粒子的密度是需要在使用微流控码技术被认为是一个关键的参数。最大p条密度可以解析（与一个CDMA电信网络的信道容量类推）取决于几个因素，如单独的传感器信号和它们之间的关系，该解码方案中，微流体装置的布局，并且电子噪声水平。根据不同的应用，可将样品稀释至达到能产生可接受的误差率的颗粒密度。 从信号处理的角度来看，从微流体码设备时间波形的解码是不计算密集使用当前系统的一个事实，即CDMA网络的移动电话的通信可以实时被解复用为证。此外，为了在微流体装置被解码的物理事件发生比在蜂窝电话通信的比特传输速率慢得多，允许使用更先进的和耗时的算法，如半导体集成电路和一个优化过程中，我们用其迭代地解决重叠SI来自传感器gnals。 两者合计，微流体码是一个通用的，可扩展的电子感测技术，可以容易地集成到各种微流体装置通过，因为它们是在芯片上处理跟踪粒子来实现定量测定。该技术是很容易实现的，因为（1）它是从硬件角度非常简单的（2）它与软光刻（3）它提供了一种直接的电子读出没有任何活性片元件直接兼容，和（4）它依赖于用于信号处理和数据解释简单的计算算法。

生物粒子如悬浮在液体中的细胞，细菌或病毒的精确检测和分析是一系列应用1，2，3的极大兴趣。在大小旗鼓相当，微流体器件提供用于此目的的独特优势，如高感光度，柔和的样本处理和控制良好的微环境，4，5，6，7。此外，微流体装置可被设计成采用流体动力学和力场的组合来被动地分馏基于各种属性8，9，10，11，12生物颗粒的异质群体。在这些设备s时，得到的颗粒分布可以用作读出，但空间信息典型地只能通过显微镜，通过其直接连接到一个实验室基础设施限制了微流体装置的实用价值。因此，一个集成的传感器，可以容易地报告粒子的时空映射，因为它们是一个微流体装置上操作，可以潜在地实现低成本，集成上实验室一个芯片装置，其对样品的移动的检测特别有吸引力，资源有限的环境。 薄膜电极已被用作微流体装置集成传感器用于各种应用13,14。电阻脉冲传感（RPS）是小颗粒在微流控通道，因为它直接从电气测量15提供了一个强大的，敏感的，高通量检测机制，整合感应特别有吸引力。在RPS，在一对电极之间的阻抗调制，浸渍在电解液中，作为检测粒子的装置。当粒子穿过的孔，尺寸的粒子的顺序，数目及电流瞬态脉冲的振幅分别用于计数和大小的颗粒。此外，该传感器的几何形状可被设计用光刻分辨率塑造电阻脉冲的波形，以提高灵敏度16，17，18，19或估计在微流体通道20的颗粒的垂直位置。 我们最近推出了可扩展和简单的复用的电阻脉冲传感技术称为微流控编码正交检测由电气传感（微流控代码）21。微码依赖于电阻脉冲传感器的互连网络，每个包含微机械以独特的，可区别的方式来调节传导电极阵列的，以便使多路复用。我们已经专门设计每一个传感器，以产生类似于在码分多路访问中使用的数字码正交的电信号22（CDMA）的电信网络中，从而使各个电阻脉冲传感器信号可以从一个单一的输出波形被唯一恢复，即使信号从不同的传感器干扰。以这种方式，我们的技术压缩颗粒的二维空间信息转换成一维的电信号，允许在一个微流体芯片上的不同位置的粒子的监测，同时保持两个器件和系统级的复杂性降至最低。 在本文中，我们提出了一个详细的协议需要使用微流控技术CODES的实验和计算方法，以及为r从它在模拟生物样品的分析使用具有代表性的结果。使用来自具有四个复用的传感器，例如一原型设备的结果来解释的技术中，我们提供了（1）的微细加工的协议来创建具有微流控码技术，（2）实验装置的说明中，包括微流体装置电子，光学，和流体硬件，（3）的计算机算法，用于解码来自不同传感器的干扰信号，以及（4）从检测和癌细胞中的微流体通道的分析的结果。我们相信，通过详细的协议这里描述，其他研究人员可以申请我们的技术为他们的研究。

<table><tbody><tr><td>98% Sulfuric Acid&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BDH Chemicals</td><td>BDH3074-3.8LP</td><td></td></tr><tr><td>30% Hydrogen Peroxide&amp;nbsp;&amp;nbsp;</td><td>BDH Chemicals</td><td>BDH7690-3</td><td></td></tr><tr><td>Trichlorosilane</td><td>Aldrich Chemistry</td><td>235725-100G</td><td></td></tr><tr><td>NR9-1500PY Negative Photoresist</td><td>Furuttex</td><td></td><td></td></tr><tr><td>Resist Developer RD6</td><td>Furuttex</td><td></td><td></td></tr><tr><td>Acetone</td><td>BDH Chemicals</td><td>BDH1101-4LP</td><td></td></tr><tr><td>SU-8 2015 Negative Photoresist</td><td>Microchem</td><td>SU8-2015</td><td></td></tr><tr><td>SU-8 Developer</td><td>Microchem</td><td>Y010200</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>3097358-1004</td><td>Sylgard 184 Silicone Elastomer Kit</td></tr><tr><td>Isopropyl Alcohol</td><td>BDH Chemicals</td><td>BDH1133-4LP</td><td></td></tr><tr><td>RPMI 1640</td><td>Corning Cellgro</td><td>10-040-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Seradigm</td><td>1500-050</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Amresco</td><td>K952-100ML</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Corning Cellgro</td><td>21-040-CM</td><td></td></tr><tr><td>PHD 22/2000 Syringe Pump</td><td>Harvard Apparatus</td><td>70-2001</td><td></td></tr><tr><td>HF2LI Lock-in Amplifier</td><td>Zurich Instrument</td><td></td><td></td></tr><tr><td>HF2TA Current Amplifier</td><td>Zurich Instrument</td><td></td><td></td></tr><tr><td>Eclipse Ti-U Microscope</td><td>Nikon Corporation</td><td></td><td></td></tr><tr><td>DS-Fi2 High-Definition Color Camera&amp;nbsp;</td><td>Nikon Corporation</td><td></td><td></td></tr><tr><td>v7.3 High-speed Camera</td><td>Phantom</td><td></td><td></td></tr><tr><td>PCIe-6361 Data Acquisition Board&amp;nbsp;</td><td>National Instruments</td><td>781050-01</td><td></td></tr><tr><td>BNC-2120 Shielded Connector Block</td><td>National Instruments</td><td>777960-01&amp;nbsp;</td><td></td></tr><tr><td>PX-250 Plasma Treatment System</td><td>Nordson MARCH&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

microfluidic platform with multiplexed electronic detection for spatial tracking of particles

生物样品的微流体处理典型地涉及以空间分馏基于感兴趣的生物特性的样品在各种力场悬浮颗粒的差动操作。对于要用作化验读数所得的空间分布，微流体装置经常受到需要具有更高的成本和减少的可移植性复杂的仪器显微镜分析。为了解决此限制，我们开发了一种集成电子传感技术用于在微流体芯片上的不同位置的粒子的多重检测。我们的技术，被称为微流控码，结合电阻脉冲感测与码分多址压缩二维空间信息转换成一维的电信号。在本文中，我们介绍了微流控技术CODES的实际演示，检测和大小培养的癌细胞分布在多个微流体通道。如由高速显微镜验证，我们的技术能够准确地分析密集细胞群体的所有电子，而不需要外部的仪器。这样，微流控代码可以潜在地使该非常适合用于生物样品的床边血糖检测低成本集成上实验室的单芯片器件。

由分布在四个微流体通道四个传感器微流体码装置， 如图1b所示。在这个系统中，每个微流体通道的横截面被设计为接近的小区的大小，以便（1）的多个细胞不能越过在平行和（2）细胞保持靠近电极提高灵敏度的电极。每个传感器被设计以产生一个唯一的7位的数字码。然后将该装置用的细胞悬浮液进行测试。对应于四个单独的传感器记录，电信号被示出在图4中相关的理想的数字码。记录的信号密切配合与理想的方波脉冲，而确实存在小的偏差。这种偏离的几个因素，包括共面的电极之间的非均匀电场的组合造成的，不同的电极对，的球状之间耦合细胞，以及在微流体通道的细胞的恒定流速。我们创建基础上，重新编码传感器信号的模板库。由所记录的信号与所有的在库中的模板相关，我们确定的模板产生的最大自相关峰值（ 图4）。作为用于微流体通道的数字码被设计成彼此正交，显性自相关峰值可以有力地在此过程中确定。使用这种方法，我们可以计算确定微流体通道中的细胞穿过时，传感器信号的持续时间，因此，细胞的流动速度。 微流体技术CODES能够解决的情况下，当多个单元同时与编码电极进行交互。当这样的重叠发生，从各个传感器的信号干扰和所得波形不能容易地与任何相关联对应于特定传感器单个模板。准确解码这种重叠信号为可靠的加工高密度的样品，其中，干扰更可能发生特别重要。要解决重叠事件，我们开发了一种基于连续干扰消除（SIC）方案24，25，其通常用于在CDMA通信网络的多用户检测的迭代算法。 图5展示了SIC算法是如何解决起因于4个重叠的细胞在四个不同的微流体通道的波形来实现。在每次迭代中，我们首先确定显性自相关峰值（ 图5a，第2列），对应于最强的干扰信号，通过与模板库的输入波形（ 图5a，1列）相关联。基于所选择的模板和叔他作为结果的自相关幅度，我们则估计最强干扰信号（ 图5a，第3列），并从输入波形中减去它。剩余的波形被传递给下一次迭代的输入。此过程持续直到残留信号与模板库的相关性没有产生明显的自相关峰值（ 图5a， 第 5行，第2图）。以下的干扰消除处理的终止，我们的所有估计的信号从每次迭代（ 图6a）将重构的波形的估计值。使用基于最小二乘逼近，以减少原有的波形和重构信号之间的均方误差的优化过程中，我们更新估计的幅度，持续时间，以及单独的传感器代码信号的相对定时（ 图6b）。我们还估计的大小在细胞中检测到基于所估计的各个传感器的信号的幅度。为了实现这一点，我们校准，使用线性回归（ 图6b）光学测定细胞大小的电信号幅度。我们从来自同时记录高速显微镜图像获得的信息的微流体码结果的比较表明，细胞的大小和速度，可以精确地测量，其验证了我们的结果（ 表1）。 图6c示出用于验证的解码结果的同时记录高速显微图像。 为了证明我们的结果的可重复性和也微流控码技术用于高通量样品处理的性能，我们分析对应&gt; 1,000个细胞的电信号。这些信号会自动在MATLAB通过运行算法解码解释上面我们的结果的准确性是通过直接比较我们的结果与同时记录高速视频光数据进行评估。我们的分析表明，从细胞的96.15％（973/1012）的电信号进行正确解码。解码非重叠和重叠细胞信号的成功率是98.71％（697分之688）及90.48％（三百一十五分之二百八十五）。 <img alt="图1" src="/files/ftp_upload/55311/55311fig1.jpg" /> 图1.四通道的微流体码装置的设计。 （ 一 ）在每个微流体通道将电极微图案，以产生一个唯一的数字代码。阻抗调制由于与电极对中流动的细胞的连续相互作用导致电脉冲。 （ 二 ）微流控代码设备的显微图像。在制造过程中，与编码表面电极的玻璃基板对准<html>用显微镜下的PDMS微流体通道。 （c）一个特写产生7位Gold序列编码表面电极的形象:"1010110"，"0111111"，"0100010"，"0011000"。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55311/55311fig1large.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/55311/55311fig2.jpg" /> 图2.微细加工工艺。 （ 一 ）PDMS微流体通道使用软光刻制造27。 （ 二 ）表面电极是使用剥离工艺制造。 （c）该最终装置的横截面示意图。的PDMS微流体通道对准并接合到与表面电极的玻璃基板。 <html> jove.com/files/ftp_upload/55311/55311fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/55311/55311fig3.jpg" /> 图3.实验装置。使用注射泵，将细胞悬浮液通过微流体码装置以恒定的流速运行。 A 400千赫的交流信号被施加到使用函数发生器参比电极。从正和负感测电极的电流信号首先被转换成使用两个阻放大器的电压信号，并使用一差分放大器彼此相减。差分的双极信号由锁定放大器萃取，然后采样成用于信号处理和解码的计算机。高速光学显微镜被用来验证和表征的目的的设备的光学记录操作。e.jove.com/files/ftp_upload/55311/55311fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55311/55311fig4.jpg" /> 图从单个传感器及其相关记录4电信号。记录的信号和它们的相互关系，给出了4个码复用的电阻脉冲传感器。传感器1（a）中 ，传感器2（b）所示，传感器3（c）和传感器4（D）被设计来产生的7位数字波形"1010110"，"0111111"，"0100010"，和"0011000"，分别。对于每个传感器，顶部的图显示了从各传感器记录的归一化信号与所述传感器被设计以产生理想的方波脉冲序列紧密匹配。对于每个传感器，下部面板显示记录的传感器信号9氏自相关和与对应于网络中的其他三个码复用传感器的信号交叉相关。在所有的情况下，自相关峰可以稳健地被识别，因为来自各个传感器的数字码被设计成彼此正交。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55311/55311fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55311/55311fig5.jpg" /> 图5.用解码连续干扰消除重叠的波形。在每次迭代中，输入波形（1列）与预组装的模板库相关，以确定导致最大相关振幅（第2列）的具体模板。采用这种特定的模板，最强的干扰信号来估计基于振幅和从相关峰（第3列）的定时信息。所估计的信号然后从原始波形中减去，从而有效地消除最强干扰由于最大小区。该过程被重复，直到没有相关峰值可被确定（ 即 ，相关系数&lt;0.5）中的残余信号。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55311/55311fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55311/55311fig6.jpg" /> 图6.解码结果分析。 （ 一 ）估计信号被细化基于优化算法，旨在获得重建，并使用最小二乘近似原记录波形之间的最佳拟合。 （b）在优化过程结束时，定时和校准信号的振幅准确地反映由高速显微镜测量的小区参数。 （ 三 ）同时记录高速显微镜图像由电气测量证实了我们的结果。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55311/55311fig6large.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> 测量类型 </td><td> - [R CH1（微米） </td><td> řCH 2（微米） </td><td> řCH3（微米） </td><td> řCH4（微米） </td><td> ΔT1（ms）的 </td><td> ΔT2（毫秒） </td><td> ΔT3（毫秒） </td></tr><tr><td>电动</td><td> 8.010 </td><td> 6.490 </td><td> 5.300 </td><td> 6.550 </td><td> 0.465 </td><td> 1.705 </td><td> 0.744 </td></tr><tr><td>光纤</td><td> 8.320 </td><td> 6.770 </td><td> 5.680 </td><td> 7.040 </td><td> 0.375 </td><td> 1.625 </td><td> 0.750 </td></tr></tbody></table> 表1. 比较图6b的电和光学测定细胞的参数。为了验证我们的估算，我们光学测定从高速显微图像的小区尺寸。不同小区之间的相对定时旋光从记录以每秒8000帧的高速视频单元之间的帧的数量来衡量。

Watch this Scientific Journal Video about Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles at JoVE.com

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

我们证明与结合电阻脉冲感测（RPS）与码分多址（CDMA）的综合表面电极网络的微流体平台，来复用在多个微流体通道的粒子的检测和大小。

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 ...

microfluidic-platform-with-multiplexed-electronic-detection-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.2K 次观看.  Georgia Institute of Technology. 我们证明与结合电阻脉冲感测（RPS）与码分多址（CDMA）的综合表面电极网络的微流体平台，来复用在多个微流体通道的粒子的检测和大小。 

视频: Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

一个单粒子为基础的微流控液力陷阱

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

科研

生物工程

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 ...

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

生物化学

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 ...

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. ...

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

通过顺序毛细管辅助组装对微生物和微粒进行图案化

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

工程学

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 ...

<meta charset="utf8"></head><body>用于单细胞因子分泌分析的微流控液滴测定

Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

<meta charset="utf8"></head><body>作者聚焦：揭示免疫反应中的多功能性和异质性

The protocol describes an advanced microfluidic platform to quantitatively measure cytokine secretion dynamics of individual human peripheral blood mononuclear cells. The platform measures up to three cytokines in parallel (IL-6, TNF&#945;, and IL-1&#946;) for each individual cell stimulated with lipopolysaccharide as an example.

微流控方法解决单个多功能细胞的同时和连续细胞因子分泌 

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. ...

与复用电子检测的粒子的空间跟踪微流体平台

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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

微流控方法解决单个多功能细胞的同时和连续细胞因子分泌

微流控方法解决单个多功能细胞的同时和连续细胞因子分泌