Name: microfluidic platform with multiplexed electronic detection for spatial tracking of particles
Uploaded: 2017-03-13T13:00:00
Description: Watch this Scientific Journal Video about Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles at JoVE.com

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><l...

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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...

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。

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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<s...

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 ...

The overall goal of this procedure is to demonstrate a microfluidic platform that combines resistive pulse sensing with code division multiple access to multiplex the detection and sizing of particles in multiple microfluidic channels. This technology, named microfluidic CODES can help realize fully integrated and truly portable labware chip devices that are well suited for the point of care testing of biological samples in resources limited settings. The main advantage of this technique is that it can electronically track spatial, temporal manipulation of particles on the microfluidic chip, eliminating the need for an external instrument such as a microscope.Our technology is compatible with soft lithography and can easily be integrated into a microphotic device where particles are fractionated to provide a direct electronic readout similar to Coulter counter. To begin construction of the microfluidic device generate a set of four, seven bit gold codes. Then design four unique electrode layouts based on the gold codes using a computer aided design, or CAD software such as AutoCAD.Finally, have the Photomasked with the designed electrode layout manufactured by a Photomask supplier. Next, soak a four inch borosilicate glass wafer in a five to one piranha solution at 120 degrees Celsius for 20 minutes. After cleaning, heat the wafer on a hot plate at 200 degrees Celsius for 20 minutes to evaporate residual water.Place the clean, dry wafer in a spin coater. Apply 2 milliliters of negative photoresist solution to the wafer and spin coat at 3000 revolutions per minute for 40 seconds. Dry the spin coated wafer on a hot plate at 150 degrees Celsius for one minute.The cover the wafer with a chrome mask in the desired electrode pattern. Expose the masked photoresist surface to 365 nanometer UV light to attain 225 millijoule per centimeter squared. The bake the exposed photoresist on a hot plate at 100 degrees Celsius for one minute.Immerse the wafer in photoresist developer for 15 seconds, then wash the pattern wafer in a gentle spray of deionized water and dry the wafer under a stream of nitrogen gas. Next, place the patterned wafer in an electron beam metal evaporator. Deposit a 20 nanometer thick chrome layer and a 80 nanometer thick gold layer onto the wafer at a rate of one Angstrom per second.Then etch the underlying photoresist by ultrasonic heating the metal coated wafer in acetone for 30 minutes at 40 kilohertz and 100%amplitude. Use a dicing saw to cut the wafer into smaller pieces as needed. To begin fabricating the microfluidic channel mold, clean and dry a four inch silicon wafer in the same way as the borosilicate wafer described previously.Place the silicon wafer in a spin coater and apply four milliliters of negative photoresist solution. Spin coat the wafer at 500 rpm for 15 seconds, then at 1, 000 rpm for 15 seconds, and finally at 3, 000 rpm for 60 seconds. Set the wafer face up on an acetone soaked clean room wipe to remove residual photoresist from the back and the edges of the wafer.Bake the wafer at 65 degrees Celsius for one minute and then at 95 degrees Celsius for two minutes. Place a chrome mask pattern for the microfluidic channels on the dry wafer. Expose the photoresist to 365 nanometer UV light at 180 millijoules per centimeter squared and then bake the wafer again at 65 and 95 degrees Celsius for one and two minutes respectively.Placed the patterned wafer in a container of photoresist developer and gently shake the container for three minutes. Rinse the developed wafer in isopropanol and dry the wafer under a stream of nitrogen gas. Bake the wafer at 200 degrees Celsius for 30 minutes, then use a profilometer to check that the pattern photoresist is uniformly thick across the wafer.Place the wafer in a vacuum desiccator along with 200 microliters of trichlorosilane in an uncovered Petri Dish. Allow the wafer to sit in the desiccator with the trichlorosilane for eight hours to silanize the wafer surface. To begin assembling the device, use general purpose clean room tape to affix the silicon wafer mold in a 150 millimeter diameter Petri Dish.Add 50 grams of a 10 to one mixture of polydimethylsiloxane prepolymer to the Petri Dish and degas the mixture in a vacuum desiccator for one hour. Cure the degassed mixture at 65 degrees Celsius for at least four hours. Use a scalpel to cut out the cured PDMS layer and then peel the cured layer off the mold with tweezers.Cut the PDMS into small pieces. Punch the inlet and outlet microfluidic channel holes with a biopsy puncher. Place the PDMS layer pattern face down on clear room tape to clean the micromachined surface.Rinse the previously prepared electrode bearing glass substrate with acetone, isopropanol, and deionized water. Dry the substrate under a stream of nitrogen gas. Place the PDMS layer and the substrate in an RF plasma generator set to 100 milliwatts with the micromachine sides facing up.Activate the micromachine surfaces in oxygen plasma for 30 seconds. Next use an optical microscope to align the pattern PDMS layer with the surface electrodes. Once aligned, allow the surfaces to come into physical contact to seal the PDMS layer onto the glass substrate.It is crucial that the coating electrode pattern on glass substrate is properly aligned with the PDMS microfluidic channels. Once aligned properly, the particle interaction with the surface electrode will generate a desired code waveform for multiplexing. Bake the assembled device at 70 degrees Celsius for five minutes, glass side down.Finally solder wires to the electrode contact pads to finish device assembly. To begin the experiment, place the microfluidic device on an optical microscope stage. Connect the device reference electrode to the signal output port of a lock-in amplifier and apply a 400 kilohertz sine wave.Connect the positive and negative sensor electrodes to two independent transimpedance amplifiers. Connect both transimpedance amplifiers to the differential voltage inputs of the lock-in amplifier with the positive sensor signal to be subtracted from the negative sensor signal. Connect the demodulator output of the lock-in amplifier to a data acquisition unit.In the data acquisition software, set a sampling rate for the lock-in amplifier output of 1 Megahertz. Set up a high speed camera to optically record the operation of the device as seen under the microscope. Draw a prepared cell suspension into a syringe.Secure the sample syringe in a syringe pump and connect the syringe to the inlet channel. Direct the outlet channel to a waste container. Use the syringe pump to drive the cell suspension through the device at a constant flow rate while recording the impedance modulation signal.After experiment's completion, process the electrical data with analysis software. Compare the processed electrical signal with images from the high speed camera to create a calibration curve for cell size. A cell suspension was flowed through a microfluidic sensor device with four unique electrode patterns derived from orthogonal sensor codes.All four sensor signals were recorded from a single electrical output. The individual sensor associated with each recorded signal was identified by correlation of the recorded sensor signals with all possible codes, which produced clearly distinguishable, autocorrelation peaks. Waveforms produced by the interfering signals from simultaneous detection of cells in all four channels were resolved with an iterative algorithm.A recorded waveform was correlated with all possible codes and the largest autocorrelation peak was identified. The corresponding individual sensor signal was reconstructed and subtracted from the input waveform. The residual signal was passed to the next iteration as the input and the process continued until the residual signal produced no autocorrelation peaks.Estimated signals were refined based on an optimization algorithm seeking the best fit between the reconstructed and the original recorded waveforms using the least squares approximation. Cell location, size, and time to cross the sensor were then determined from the channel number, amplitude, duration, and relative timing of the estimated sensor signals. The procedure was validated by comparison of the electrical signals with optical measurements from the high speed camera.Once mastered, this technique is very easy to implement, because it is very simple from a hardware perspective. It has no active on-chip component. It is directly compatible with soft lithography and the signal processing relies on a simple computational algorithm.Following this protocol you can fabricate microfluidic chips with code based multiplex electrical sensors and decode electrical signals for bioanalytical measurement. This versatile, scalable, electronic sensing technology, can be readily integrated into various microfluidic devices to realize quantitative assays by spatial temporally tracking particles as they processed on the chip. After watching this vide, you should have a good understanding of how to design, fabricate, and implement a microfluidic CODES technology.

Research

JoVE Journal

Bioengineering

A Microfluidic-based Hydrodynamic Trap for Single Particles

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

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 ...

Bridging the Bio-Electronic Interface with Biofabrication

生物电子界面衔接与Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

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

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. ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

影像学在两层微流控平台拟南芥幼苗的根头发形态

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...