Integrated circuit/microfluidic chip to programmably trap and move cells and droplets with dielectrophoresis

We present an integrated circuit/microfluidic chip that traps and moves individual living biological cells and chemical droplets along programmable paths using dielectrophoresis (DEP). Our chip combines the biocompatibility of microfluidics with the programmability and complexity of integrated circuits (ICs). The chip is capable of simultaneously and independently controlling the location of thousands of dielectric objects, such as cells and chemical droplets. The chip consists of an array of 128 x 256 pixels, 11 x 11 microm(2) in size, controlled by built-in SRAM memory; each pixel can be energized by a radio frequency (RF) voltage of up to 5 V(pp). The IC was built in a commercial foundry and the microfluidic chamber was fabricated on its top surface at Harvard. Using this hybrid chip, we have moved yeast and mammalian cells through a microfluidic chamber at speeds up to 30 microm sec(-1). Thousands of cells can be individually trapped and simultaneously positioned in controlled patterns. The chip can trap and move pL droplets of water in oil, split one droplet into two, and mix two droplets into one. Our IC/microfluidic chip provides a versatile platform to trap and move large numbers of cells and fluid droplets individually for lab-on-a-chip applications.     

阵列式对电极介电电泳芯片及其用于细胞分离富集研究

基于介电电泳原理, 设计并制作了一种新型的能够用于细胞分离和富集的微流控介电电泳芯片. 该芯片由沉积有金电极的石英基片和带有微管道的聚二甲基硅氧烷(PDMS)盖片组成. 通过在管道底部布置间距不同的对电极阵列, 增大了正介电电泳力在管道中的有效作用范围, 能够在降低施加电压的同时, 实现对流动体系中细胞样品的捕获. 在3 V和3 MHz条件下, 该DEP芯片对人血红细胞的捕获效率达到83%; 进一步通过将肝癌细胞捕获在芯片电极上可实现对红细胞和肝癌细胞混合样品的分离, 在5 V和400 kHz条件下对肝癌细胞的捕获效率达到86%.     

Dielectrophoresis-based cellular microarray chip for anticancer drug screening in perfusion microenvironments

We present a dielectrophoresis (DEP)-based cellular microarray chip for cell-based anticancer drug screening in perfusion microenvironments. Human breast cancer cells, MCF7, were seeded into the chip and patterned via DEP forces onto the planar interdigitated ring electrode (PIRE) arrays. Roughly, only one third of the cell amount was required for the chip compared to that for a 96-well plate control. Drug concentrations (cisplatin or docetaxel) were stably generated by functional integration of a concentration gradient generator (CGG) and an anti-crosstalk valve (ACV) to treat cells for 24 hours. Cell viability was quantified using a dual staining method. Results of cell patterning show substantial uniformity of patterned cells (92 ± 5 cells per PIRE). Furthermore, after 24 hour drug perfusion, no statistical significance in dose-responses between the chip and the 96-well plate controls was found. The IC(50) value from the chip also concurred with the values from the literature. Moreover, the perfusion culture exhibited reproducibility of drug responses of cells on different PIREs in the same chamber. The chip would enable applications where cells are of limited supply, and supplement microfluidic perfusion cultures for clinical practices.     

A flow-through microfluidic chip for continuous dielectrophoretic separation of viable and non-viable human T-cells

Effective methods for rapid sorting of cells according to their viability are critical in T cells based therapies to prevent any risk to patients. In this context, we present a novel microfluidic device that continuously separates viable and non-viable T-cells according to their dielectric properties. A dielectrophoresis (DEP) force is generated by an array of castellated microelectrodes embedded into a microfluidic channel with a single inlet and two outlets; cells subjected to positive DEP forces are drawn toward the electrodes array and leave from the top outlet, those subjected to negative DEP forces are repelled away from the electrodes and leave from the bottom outlet. Computational fluid dynamics is used to predict the device separation efficacy, according to the applied alternative current (AC) frequency, at which the cells move from/to a negative/positive DEP region and the ionic strength of the suspension medium. The model is used to support the design of the operational conditions, confirming a separation efficiency, in terms of purity, of 96% under an applied AC frequency of 1.5 × 10⁶ Hz and a flow rate of 20 μl/h. This work represents the first example of effective continuous sorting of viable and non-viable human T-cells in a single-inlet microfluidic chip, paving the way for lab-on-a-chip applications at the point of need.     

Multistep manipulations of poly(methyl‐methacrylate) submicron particles using dielectrophoresis

A microfluidic chip for multistep manipulations of PMMA submicron particles (PMMA‐SMPs) based on dielectrophoresis (DEP) has been developed that includes four main functions of focusing, guiding, trapping, and releasing the SMPs. The structure of the DEP chip consists of a top electrode made of indium tin oxide, a flow chamber formed by optically clear adhesive tape and bottom electrodes with different patterns for different purposes. The bottom electrodes can be divided into three parts: a fish‐bone‐type electrode array that provides the positive DEP force for focusing the suspended nanoparticles (NPs) near the inlet in the flow chamber; the second is for switching and guiding the focused NPs along the electrode surface to the target area, like a flow passing along a virtual channel; and a trapping electrode in the downstream for trapping and releasing the guided NPs. According to the simulation and experimental results, NPs can be aligned along the electrode of the focusing electrode and guided toward the target electrode by means of a positive DEP force between the top and bottom electrodes, with the effects of Brownian motion and Stokes force. In order to demonstrate the sequence of DEP manipulations, a PMMA‐NP suspension is introduced to the DEP chip; the size of the PMMA‐SMPs is about 300 nm. Furthermore, a LabVIEW program developed for sequence control of the AC signals for the multistep manipulations. Consequently, the DEP chip provides an excellent platform technology for the multistep manipulation of SMPs.     

Patterned cell culture inside microfluidic devices

This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern cell-adhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to well-controlled fluidic microenvironment.     

Construction of Tumor Tissue Array on An Open-Access Microfluidic Chip

An open-access microfluidic chip which enabled automatic cell distribution and complex multi-step operations was developed. The microfluidic chip featured a key structure in which a nanoporous membrane was sandwiched by a cell culture chamber array layer and a corresponding media reservoir array layer. The microfluidic approach took advantage of the characteristics of nanoporous membrane. On one side, this membrane permitted the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution. On the other side, it allowed diffusion-based media exchange and thus, mimicked the endothelial layer. In synergy with a liquid transferring platform, the open-access microfluidic system enabled complex multi-step operations involving medium exchange, drug treatment, and cell viability testing. By using this microfluidic protocol, a 10 × 10 tissue arrays was constructed in 90 s, followed by schedule-dependent drug testing. Morphological and immunohistochemical assays results indicated that the resultant tumor tissue was faithful to that in vivo. Drug testing assays showed that the microfluidic tissue array promised multi-step cell assays under biomimetic microenvironment, thus providing an advantageous tool for cell research.     

Quantitative modeling of dielectrophoretic traps

We present quantitative modeling software for simulating multiple forces acting on a single particle in a microsystem. In this paper, we focus on dielectrophoretic (DEP) trapping of single cells against fluid flow. The software effectively models the trapping behavior for a range of particles including beads, mammalian cells, viruses, and bacteria. In addition, the software can be used to reveal useful information about the DEP traps - such as multipolar DEP force effects, trap size-selectivity, and effects from varying the flow chamber height. Our modeling software thus serves as a predictive tool, enabling the design of novel DEP traps with superior performance over existing trap geometries. In addition, the software can evaluate a range of trap dimensions to determine the effects on trapping behavior, thus optimizing the trap geometry before it is even fabricated. The software is freely available to the scientific community at: .     

Dielectrophoretic separation of microalgae cells in ballast water in a microfluidic chip

The composition of the ship's ballast water is complex and contains a large number of microalgae cells, bacteria, microplastics, and other microparticles. To increase the accuracy and efficiency of detection of the microalgae cells in ballast water, a new microfluidic chip for continuous separation of microalgae cells based on alternating current dielectrophoresis was proposed. In this microfluidic chip, one piece of 3‐dimensional electrode is embedded on one side and eight discrete electrodes are arranged on the other side of the microchannel. An insulated triangular structure between electrodes is designed for increasing the inhomogeneity of the electric field distribution and enhancing the dielectrophoresis (DEP) force. A sheath flow is designed to focus the microparticles near the electrode, so as to increase the suffered DEP force and improve separation efficiency. To demonstrate the performance of the microfluidic separation chip, we developed two species of microalgae cells (Platymonas and Closterium) and a kind of microplastics to be used as test samples. Analyses of the related parameters and separation experiments by our designed microfluidic chip were then conducted. The results show that the presented method can separate the microalgae cells from the mixture efficiently, and this is the first time to separate two or more species of microalgae cells in a microfluidic chip by using negative and positive DEP force simultaneously, and moreover it has some advantages including simple operation, high efficiency, low cost, and small size and has great potential in on‐site pretreatment of ballast water.     

Microfluidic lithography of SAMs on gold to create dynamic surfaces for directed cell migration and contiguous cell cocultures

A straightforward, flexible, and inexpensive method to create patterned self-assembled monolayers (SAMs) on gold using microfluidics-microfluidic lithography-has been developed. Using a microfluidic cassette, alkanethiols were rapidly patterned on gold surfaces to generate monolayers and mixed monolayers. The patterning methodology is flexible and, by controlling the solvent conditions and thiol concentration, permeation of alkanethiols into the surrounding PDMS microfluidic cassette can be advantageously used to create different patterned feature sizes and to generate well-defined SAM surface gradients with a single microfluidic chip. To demonstrate the utility of microfluidic lithography, multiple cell experiments were conducted. By patterning cell adhesive regions in an inert background, a combination of selective masking of the surface and centrifugation achieved spatial and temporal control of patterned cells, enabling the design of both dynamic surfaces for directed cell migration and contiguous cocultures. Cellular division and motility resulted in directed, dynamic migration, while the centrifugation-aided seeding of a second cell line produced contiguous cocultures with multiple sites for heterogeneous cell-cell interactions.     

用于细胞三维培养的集成微柱阵列的微流控芯片设计与验证

设计并验证了一种用于细胞三维培养的集成微柱阵列的微流控芯片.芯片由一片聚二甲基硅氧烷(PDMS)沟道片和一片玻璃盖片组成, 在PDMS沟道片上集成了一个由两排微柱阵列围成的细胞培养室和两条用于输送培养基的侧沟道.微柱间距直接影响了芯片的使用性能, 是整个芯片设计的关键.基于数值模拟和实验验证, 本研究对微柱间距进行了优化设计.优化后的微流控芯片可以很好地实现细胞与细胞外基质模拟材料混合液的稳定注入、培养基中营养物质向培养室内的快速扩散和细胞代谢物的及时排出.在芯片上进行了神经干细胞的三维培养, 证明了芯片上构建的细胞体外微环境的稳定性.     

Fast immobilization of probe beads by dielectrophoresis-controlled adhesion in a versatile microfluidic platform for affinity assay

The use of probe beads for lab-on-chip affinity assays is very interesting from a practical point of view. It is easier to handle and trap beads than molecules in microfluidic systems. We present a method for the immobilization of probe beads at defined areas on a chip using dielectrophoresis (DEP)-controlled adhesion. The method is fast, i.e., it takes between 10 and 120 s--depending on the protocol--to functionalize a chip surface at defined areas. The method is versatile, i.e., it works for beads with different types of probe molecule coatings. The immobilization is irreversible, i.e., the retained beads are able to withstand high flow velocities in a flow-through device even after the DEP voltage is turned off, thus allowing the use of conventional high-conductivity analyte buffers in the following assay procedure. We demonstrate the on-chip immobilization of fluorescent beads coated with biotin, protein A, and goat-antimouse immunoglobulin G (IgG). The number of immobilized beads at an electrode array can be determined from their fluorescence signal. Further, we use this method to demonstrate the detection of streptavidin and mouse IgG. Finally, we demonstrate the feasibility of the parallel detection of different analyte molecules on the same chip.     

On‐line separation of bacterial cells by carbon‐electrode dielectrophoresis

Dielectrophoresis (DEP) represents a powerful approach to manipulate and study living cells. Hitherto, several approaches have used 2‐D DEP chips. With the aim to increase sample volume, in this study we used a 3‐D carbon‐electrode DEP chip to trap and release bacterial cells. A continuous flow was used to plug an Escherichia coli cell suspension first, to retain cells by positive DEP, and thereafter to recover them by washing with peptone water washing solution. This approach allows one not only to analyze DEP behavior of living cells within the chip, but also to further recover fractions containing DEP‐trapped cells. Bacterial concentration and flow rate appeared as critical parameters influencing the separation capacity of the chip. Evidence is presented demonstrating that the setup developed in this study can be used to separate different types of bacterial cells.     

PCR-based detection in a micro-fabricated platform

We present a novel, on-chip system for the electrokinetic capture of bacterial cells and their identification using the polymerase chain reaction (PCR). The system comprises a glass-silicon platform with a set of micro-channels, -chambers, and -electrodes. A platinum thin film resistor, placed in the proximity of the chambers, is used for temperature monitoring. The whole chip assembly is mounted on a Printed Circuit Board (PCB) and wire-bonded to it. The PCB has an embedded heater that is utilized for PCR thermal cycle and is controlled by a Lab-View program. Similar to our previous work, one set of electrodes on the chip inside the bigger chamber (0.6 microl volume) is used for diverting bacterial cells from a flowing stream into to a smaller chamber (0.4 nl volume). A second set of interdigitated electrodes (in smaller chamber) is used to actively trap and concentrate the bacterial cells using dielectrophoresis (DEP). In the presence of the DEP force, with the cells still entrapped in the micro-chamber, PCR mix is injected into the chamber. Subsequently, PCR amplification with SYBR Green detection is used for genetic identification of Listeria monocytogenes V7 cells. The increase in fluorescence is recorded with a photomultiplier tube module mounted over an epifluorescence microscope. This integrated micro-system is capable of genetic amplification and identification of as few as 60 cells of L. monocytogenes V7 in less than 90 min, in 600 nl volume collected from a sample of 10(4) cfu ml(-1). Specificity trials using various concentrations of L. monocytogenes V7, Listeria innocua F4248, and Escherichia coli O157:H7 were carried out successfully using two different primer sets specific for a regulatory gene of L. monocytogenes, prfA and 16S rRNA primer specific for the Listeria spp., and no cross-reactivity was observed.     

Multiphase electropatterning of cells and biomaterials

Tissues formed by cells encapsulated in hydrogels have uses in biotechnology, cell-based assays, and tissue engineering. We have previously presented a 3D micropatterning technique that rapidly localizes live cells within hydrogels using dielectrophoretic (DEP) forces, and have demonstrated the ability to modulate tissue function through the control of microscale cell architecture. A limitation of this method is the requirement that a single biomaterial must simultaneously harbor biological properties that support cell survival and function and material properties that permit efficient dielectrophoretic patterning. Here, we resolve this issue by forming multiphase tissues consisting of microscale tissue sub-units in a 'local phase' biomaterial, which, in turn, are organized by DEP forces in a separate, mechanically supportive 'bulk phase' material. We first define the effects of medium conductivity on the speed and quality of DEP cell patterning. As a case study, we then produce multiphase tissues with microscale architecture that combine high local hydrogel conductivity for enhanced survival of sensitive liver progenitor cells with low bulk conductivity required for efficient DEP micropatterning. This approach enables an expanded range of studies examining the influence of 3D cellular architecture on diverse cell types, and in the future may improve the biological function of inhomogeneous tissues assembled from a variety of modular tissue sub-units.     

A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array

We present a high aspect ratio microfluidic device for culturing cells inside an array of microchambers with continuous perfusion of medium. The device was designed to provide a potential tool for cost-effective and automated cell culture. The single unit of the array consists of a circular microfluidic chamber 40 microm in height surrounded by multiple narrow perfusion channels 2 microm in height. The high aspect ratio (approximately 20) between the microchamber and the perfusion channels offers advantages such as localization of the cells inside the microchamber as well as creating a uniform microenvironment for cell growth. Finite element methods were used to simulate flow profile and mass transfer of the device. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C and was grown to confluency. The microfluidic cell culture array could potentially offer an affordable platform for a wide range of applications in high throughput cell-based screening, bioinformatics, synthetic biology, quantitative cell biology, and systems biology.     

单细胞分析的研究

单细胞分析是分析化学、生物学和医学之间渗透发展形成的跨学科前沿领域。近年来,毛细管电泳及微流控芯片用于单细胞分析已取得显著进展,特别表现在微流控芯片用于细胞的培养、分选、操纵、定位、分离及检测细胞的组分,实时监测细胞释放,及高通量阵列检测等方面。芯片的单元操作可根据需要灵活组合,显示出其独特的优点。本文重点介绍作者研究组的工作,并对近三年来国内外在毛细管电泳及芯片毛细管电泳用于单细胞分析的新进展进行评论。最后从毛细管电泳与微流控芯片、微流控芯片与细胞界面以及量子点用于探测活细胞等方面,展望了单细胞分析的发展前景。     

Frequency discretization in dielectrophoretic assisted cell sorting arrays to isolate neural cells

We present an automated dielectrophoretic assisted cell sorting (DACS) device for dielectric characterization and isolation of neural cells. Dielectrophoretic (DEP) principles are often used to develop cell sorting techniques. Here we report the first statistically significant neuronal sorting using DACS to enrich neurons from a heterogeneous population of mouse derived neural stem/progenitor cells (NSPCs) and neurons. We also study the dielectric dispersions within a heterogeneous cell population using a Monte-Carlo (MC) simulation. This simulation model explains the trapping behavior of populations as a function of frequency and predicts sorting efficiencies. The platform consists of a DEP electrode array with three multiplexed trapping regions that can be independently activated at different frequencies. A novel microfluidic manifold enables cell sorting by trapping and collecting cells at discrete frequency bands rather than single frequencies. The device is used to first determine the percentage of cells trapped at these frequency bands. With this characterization and the MC simulation we choose the optimal parameters for neuronal sorting. Cell sorting experiments presented achieve a 1.4-fold neuronal enrichment as predicted by our model.     

Cross-scale electric manipulations of cells and droplets by frequency-modulated dielectrophoresis and electrowetting

Two important electric forces, dielectrophoresis (DEP) and electrowetting-on-dielectric (EWOD), are demonstrated by dielectric-coated electrodes on a single chip to manipulate objects on different scales, which results in a dielectrophoretic concentrator in an EWOD-actuated droplet. By applying appropriate electric signals with different frequencies on identical electrodes, EWOD and DEP can be selectively generated on the proposed chip. At low frequencies, the applied voltage is consumed mostly in the dielectric layer and causes EWOD to pump liquid droplets on the millimetre scale. However, high frequency signals establish electric fields in the liquid and generate DEP forces to actuate cells or particles on the micrometre scale inside the droplet. For better performance of EWOD and DEP, square and strip electrodes are designed, respectively. Mammalian cells (Neuro-2a) and polystyrene beads are successfully actuated by a 2 MHz signal in a droplet by positive DEP and negative DEP, respectively. Droplet splitting is achieved by EWOD with a 1 kHz signal after moving cells or beads to one side of the droplet. Cell concentration, measured by a cell count chamber before and after experiments, increases 1.6 times from 8.6 x 10(5) cells ml(-1) to 1.4 x 10(6) cells ml(-1) with a single cycle of positive DEP attraction. By comparing the cutoff frequency of the voltage drop in the dielectric layer and the cross-over frequency of Re(fCM) of the suspended particles, we can estimate the frequency-modulated behaviors between EWOD, positive DEP, and negative DEP. A proposed weighted Re(fCM) facilitates analysis of the DEP phenomenon on dielectric-coated electrodes.     

Multiplexed proteomic sample preconcentration device using surface-patterned ion-selective membrane

In this paper, we report a new method of fabricating a high-throughput protein preconcentrator in poly(dimethylsiloxane) (PDMS) microfluidic chip format. We print a submicron thick ion-selective membrane on the glass substrate by using standard patterning techniques. By simply plasma-bonding a PDMS microfluidic device on top of the printed glass substrate, we can integrate the ion-selective membrane into the device and rapidly prototype a PDMS preconcentrator without complicated microfabrication and cumbersome integration processes. The PDMS preconcentrator shows a concentration factor as high as approximately 10(4) in 5 min. This printing method even allows fabricating a parallel array of preconcentrators to increase the concentrated sample volume, which can facilitate an integration of our microfluidic preconcentrator chip as a signal enhancing tool to various detectors such as a mass spectrometer.