T cell chemotaxis in a simple microfluidic device

This paper describes the use of a simple microfluidic device for studying T cell chemotaxis. The microfluidic device is fabricated in poly(dimethylsiloxane) (PDMS) using soft-lithography and consists of a "Y" type fluidic channel. Solutions are infused into the device by syringe pumps and generate a concentration gradient in the channel by diffusion. We show that the experimentally measured gradient profiles agree nicely with theoretical predictions and the gradient is stable in the observation region for cell migration. Using this device, we demonstrate robust chemotaxis of human T cells in response to single and competing gradients of chemokine CCL19 and CXCL12. Because of the simplicity of the device, it can flexibly control gradient generation in space and time, and would allow generation of multiple gradient conditions in a single chip for highly parallel chemotaxis experimentation. Visualization of T cell chemotaxis has previously been limited to studies in 3D matrices or under agarose assays, which do not allow precise control or variation in conditions. Acknowledging the importance of lymphocyte homing in the adaptive immune response, the ability to study T cell chemotaxis in microfluidic devices offers a new approach for investigating lymphocyte migration and chemotaxis in vitro.     

Effects of flow and diffusion on chemotaxis studies in a microfabricated gradient generator

An understanding of chemotaxis at the level of cell-molecule interactions is important because of its relevance in cancer, immunology, and microbiology, just to name a few. This study quantifies the effects of flow on cell migration during chemotaxis in a microfluidic device. The chemotaxis gradient within the device was modeled and compared to experimental results. Chemotaxis experiments were performed using the chemokine CXCL8 under different flow rates with human HL60 promyelocytic leukemia cells expressing a transfected CXCR2 chemokine receptor. Cell trajectories were separated into x and y axis components. When the microchannel flow rates were increased, cell trajectories along the x axis were found to be significantly affected (p < 0.05). Total migration distances were not affected. These results should be considered when using similar microfluidic devices for chemotaxis studies so that flow bias can be minimized. It may be possible to use this effect to estimate the total tractile force exerted by a cell during chemotaxis, which would be particularly valuable for cells whose tractile forces are below the level of detection with standard techniques of traction-force microscopy.     

3D Insulator‐based dielectrophoresis using DC‐biased,AC electric fields for selective bacterial trapping

Insulator‐based dielectrophoresis (iDEP) is a well‐known technique that harnesses electric fields for separating, moving, and trapping biological particle samples. Recent work has shown that utilizing DC‐biased AC electric fields can enhance the performance of iDEP devices. In this study, an iDEP device with 3D varying insulating structures analyzed in combination with DC biased AC fields is presented for the first time. Using our unique reactive ion etch lag, the mold for the 3D microfluidic chip is created with a photolithographic mask. The 3D iDEP devices, whose largest dimensions are 1 cm long, 0.18 cm wide, and 90 μm deep are then rapidly fabricated by curing a PDMS polymer in the glass mold. The 3D nature of the insulating microstructures allows for high trapping efficiency at potentials as low as 200 Vpp. In this work, separation of Escherichia coli from 1 μm beads and selective trapping of live Staphylococcus aureus cells from dead S. aureus cells is demonstrated. This is the first reported use of DC‐biased AC fields to selectively trap bacteria in 3D iDEP microfluidic device and to efficiently separate particles where selectivity of DC iDEP is limited.     

Bacterial inactivation via microfluidic electroporation device with insulating micropillars

Electroporation is a promising method to inactivate cells and it has wide applications in medical science, biology and environmental health. Here, we investigate the bacteria inactivation performance of two different microfluidic electroporation devices with rhombus and circular micropillars used for generating locally enhanced electric field strength. Experiments are carried out to characterize the inactivation performance (i.e., the log removal efficiency) of two types of bacteria: Escherichia coli (E. coli, gram-negative) and Enterococcus faecalis (E. faecalis, gram-positive) in these two microfluidic devices. We find that under the same applied electric field, the device with rhombus micropillars performs better than the device with circular micropillars for both E. coli and E. faecalis. Numerical simulations show that due to the corner-induced singularity effect, the maximum electric field enhancement is higher in the device with rhombus micropillars than that in the device with circular micropillars. We also study the effects of DC and AC electric fields and flowrate. Our experiments demonstrate that the use of the DC field achieves higher log removal efficiencies than the use of AC field.     

Microfluidic device embedding electrodes for dielectrophoretic manipulation of cells‐A review

Microfluidic device embedding electrodes realizes cell manipulation with the help of dielectrophoresis. Cell manipulation is an important technology for cell sorting and cell population purification. Till now, the theory of dielectrophoresis has been greatly developed. Microfluidic devices with various arrangements of electrodes have been reported from the beginning of the single non‐uniform electric field to the later multiple physical fields. This paper reviews the research status of microfluidic device embedding electrodes for cell manipulation based on dielectrophoresis. Firstly, the working principle of dielectrophoresis is explained. Next, cell manipulation approaches based on dielectrophoresis are introduced. Then, different types of electrode arrangements in the microfluidic device for cell manipulation are discussed, including planar, multilayered and microarray dot electrodes. Finally, the future development trend of the dielectrophoresis with the help of microfluidic devices is prospected. With the rapid development of microfluidic technology, in the near future, high precision, high throughput, high efficiency, multifunctional, portable, economical and practical microfluidic dielectrophoresis will be widely used in the fields of biology, medicine, agriculture and so on.     

基于微流控芯片的细菌趋化性检测研究进展

细菌趋化性是指有运动能力的细菌对环境化学物质梯度产生响应,趋向某些化学诱导剂或避开某些化学驱避剂的移动行为,是微生物适应环境变化而生存的一种基本属性.研究细菌趋化性对于利用细菌治理环境、控制病原菌侵染机体以及开发微生物工业项目等方面都具有重要意义.微流控芯片可以实现对细菌趋化性的定性与定量检测,与传统的检测方法相比,可以更好地对细菌的微环境进行控制,有较高的灵敏度.近年来,基于微流控技术检测细菌趋化性研究得到了飞速发展.本文从微流控芯片的结构、工作方式及主要应用3个方面对近年出现的微流控趋化性检测装置进行了介绍和评述.     

A cell electro‐rotation micro‐device using polarized cells as electrodes

Cell rotation is widely required in various fields as an important technique for single cell manipulation. Usually, the electro‐rotational manipulation of single cells by dielectrophoresis technologies requires at least three electrodes to generate rotating electric fields which induce cells to rotate. Here, we present a novel microfluidic chip capable of rotating single cell using only two planar electrodes by taking polarized cells as the extra electrodes with phase‐shifted signal. To demonstrate this idea, we configured two parallel and planar electrodes as basic dielectrophoresis elements and placed trenches above these electrodes to attract cells, which were in turn polarized to be electrodes. Through simulation, we confirmed the functional structure of the device works well to generate proper rotating electric fields for cell rotation. Through experiment, we successfully demonstrated controlled electro‐rotation of HeLa and HepaRG cells. The novel electro‐rotation mechanism not only simplifies the micro‐device structure but also reduces the complexity of single cell rotation operation which will be a benefit to the potential users.     

Endothelial cell polarization and chemotaxis in a microfluidic device

The directed migration of endothelial cells is an early and critical step in angiogenesis, or new blood vessel formation. In this study, the polarization and chemotaxis of human umbilical vein endothelial cells (HUVEC) in response to quantified gradients of vascular endothelial growth factor (VEGF) were examined. To accomplish this, a microfluidic device was designed and fabricated to generate stable concentration gradients of biomolecules in a cell culture chamber while minimizing the fluid shear stress experienced by the cells. Finite element simulation of the device geometry produced excellent agreement with the observed VEGF concentration distribution, which was found to be stable across multiple hours. This device is expected to have wide applicability in the study of shear-sensitive cells such as HUVEC and non-adherent cell types as well as in the study of migration through three-dimensional matrices. HUVEC were observed to chemotax towards higher VEGF concentrations across the entire range of concentrations studied (18-32 ng mL(-1)) when the concentration gradient was 14 ng mL(-1) mm(-1). In contrast, shallow gradients (2 ng mL(-1) mm(-1)) across the same concentration range were unable to induce HUVEC chemotaxis. Furthermore, while all HUVEC exposed to elevated VEGF levels (both in steep and shallow gradients) displayed an increased number of filopodia, only chemotaxing HUVEC displayed an asymmetric distribution of filopodia, with enhanced numbers of protrusions present along the leading edge. These results suggest a two-part requirement to induce VEGF chemotaxis: the VEGF absolute concentration enhances the total number of filopodia extended while the VEGF gradient steepness induces filopodia localization, cell polarization, and subsequent directed migration.     

Hydrogel microfluidic co‐culture device for photothermal therapy and cancer migration

We developed the photo‐crosslinkable hydrogel microfluidic co‐culture device to study photothermal therapy and cancer cell migration. To culture MCF7 human breast carcinoma cells and metastatic U87MG human glioblastoma in the microfluidic device, we used 10 w/v% gelatin methacrylate (GelMA) hydrogels as a semi‐permeable physical barrier. We demonstrated the effect of gold nanorod on photothermal therapy of cancer cells in the microfluidic co‐culture device. Interestingly, we observed that metastatic U87MG human glioblastoma largely migrated toward vascular endothelial growth factor (VEGF)‐treated GelMA hydrogel‐embedding microchannels. The main advantage of this hydrogel microfluidic co‐culture device is to simultaneously analyze the physiological migration behaviors of two cancer cells with different physiochemical motilities and study gold nanorod‐mediated photothermal therapy effect. Therefore, this hydrogel microfluidic co‐culture device could be a potentially powerful tool for photothermal therapy and cancer cell migration applications.     

Mesenchymal‐Mode Migration Assay and Antimetastatic Drug Screening with High‐Throughput Microfluidic Channel Networks

Increasing evidence shows that activated mesenchymal migration is a key process of the metastatic cascade. Cancer cells usually gain such migratory capability through an epithelial‐to‐mesenchymal transition. Herein we present a high‐throughput microfluidic device with 3120 microchambers to specifically monitor mesenchymal migration. Through imaging of the whole chip and statistical analysis, we can evaluate the two key factors of velocity and percentage related to cell migratory capacity at different cell densities in culture. We also used the device to screen antimetastatic drugs for their inhibition of mesenchymal migration and prevention of metastatic malignancy. This device will provide an excellent platform for biologists to gain a better understanding of cancer metastasis.     

Microfluidic device for analyzing preferential chemotaxis and chemoreceptor sensitivity of bacterial cells toward carbon sources

We present a novel microfluidic device that enables high sensitive analyses of the chemotactic response of motile bacterial cells (Escherichia coli) that swim toward a preferred nutrient by sorting and concentrating them. The device consists of the Y-shaped microchannel that has been widely used in chemotaxis studies to attract cells toward a high concentration and a concentrator array integrated with arrowhead-shaped ratchet structures beside the main microchannel to trap and accumulate them. Since the number of accumulated cells in the concentrator array continuously increases with time, the device makes it possible to increase the sensitivity of detecting chemotactic responses of the cells about 10 times greater than Y-shaped channel devices in 60 min. In addition, the device can characterize the relative chemotactic sensitivity of chemoreceptors to chemoeffectors by comparing the number of cells in the concentrator array at different distances from the channel junction. Since the device allows the analysis of both the chemotactic responses and the sensitivity of chemoreceptors with high resolution, we believe that not only can the device be broadly used for various microbial chemotaxis assays but it also can further the advancement of microbiology and even synthetic biology.     

The potential of autofluorescence for the detection of single living cells for label-free cell sorting in microfluidic systems

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.     

Controlled generation of droplets using an electric field in a flow-focusing paper-based device

Droplet-based microfluidics is a modular platform in high-throughput single-cell and small sample analyses. However, this droplet microfluidic system was widely fabricated using soft lithography or glass capillaries, which is expensive and technically demanding for various applications, limiting use in resource-poor settings. Besides, the variation in droplet size is also restricted due to the limitations on the operating forces that the paper-based platform is able to withstand. Herein, we develop a fully integrated paper-based droplet microfluidic platform for conducting droplet generation and cell encapsulation in independent aqueous droplets dispersed in a carrier oil by incorporating electric fields. Through imposing an electric field, the droplet size would decrease with increasing the electric field and smaller droplets can be produced at high applied voltage. The droplet diameter can be adjusted by the ratio of inner and outer flow velocities as well as the applied electric field. We also demonstrated the proof of concept encapsulation application of our paper device by encapsulating yeast cells under an electric field. Using a simple wax printing method, carbon electrodes can be integrated on the paper. The integrated paper-based microfluidic platform can be fabricated easily and conducted outside of centralized laboratories. This microfluidic system shows great potential in drug and cell investigations by encapsulating cells in resource-limited environments.     

Selective trapping of single mammalian breast cancer cells by insulator-based dielectrophoresis

The trapping or immobilization of individual cells at specific locations in microfluidic platforms is essential for single cell studies, especially those requiring cell stimulation and downstream analysis of cellular content. Selectivity for individual cell types is required when mixtures of cells are analyzed in heterogeneous and complex matrices, such as the selection of metastatic cells within blood samples. Here, we demonstrate a microfluidic device based on direct current (DC) insulator-based dielectrophoresis (iDEP) for selective trapping of single MCF-7 breast cancer cells from mixtures with both mammalian peripheral blood mononuclear cells (PBMC) as well MDA-MB-231 as a second breast cancer cell type. The microfluidic device has a teardrop iDEP design optimized for the selective capture of single cells based on their differential DEP behavior under DC conditions. Numerical simulations adapted to experimental device geometries and buffer conditions predicted the trapping condition in which the dielectrophoretic force overcomes electrokinetic forces for MCF-7 cells, whereas PBMCs were not trapped. Experimentally, selective trapping of viable MCF-7 cells in mixtures with PBMCs was demonstrated in good agreement with simulations. A similar approach was also executed to demonstrate the selective trapping of MCF-7 cells in a mixture with MDA-MB-231 cells, indicating the selectivity of the device for weakly invasive and highly invasive breast cancer cells. The DEP studies were complemented with cell viability tests indicating acceptable cell viability over the course of an iDEP trapping experiment.

Recent applications of AC electrokinetics in biomolecular analysis on microfluidic devices

AC electrokinetics is a generic term that refers to an induced motion of particles and fluids under nonuniform AC electric fields. The AC electric fields are formed by application of AC voltages to microelectrodes, which can be easily integrated into microfluidic devices by standard microfabrication techniques. Moreover, the magnitude of the motion is large enough to control the mass transfer on the devices. These advantages are attractive for biomolecular analysis on the microfluidic devices, in which the characteristics of small space and microfluidics have been mainly employed. In this review, I describe recent applications of AC electrokinetics in biomolecular analysis on microfluidic devices. The applications include fluid pumping and mixing by AC electrokinetic flow, and manipulation of biomolecules such as DNA and proteins by various AC electrokinetic techniques. Future prospects for highly functional biomolecular analysis on microfluidic devices with the aid of AC electrokinetics are also discussed.     

Direct current insulator-based dielectrophoretic characterization of erythrocytes: ABO-Rh human blood typing

A microfluidic platform developed for quantifying the dependence of erythrocyte (red blood cell, RBC) responses by ABO-Rh blood type via direct current insulator dielectrophoresis (DC-iDEP) is presented. The PDMS DC-iDEP device utilized a 400 x 170?μm2 rectangular insulating obstacle embedded in a 1.46-cm long, 200-μm wide inlet channel to create spatial non-uniformities in direct current (DC) electric field density realized by separation into four outlet channels. The DC-iDEP flow behaviors were investigated for all eight blood types (A+, A-, B+, B-, AB+, AB-, O+, O-) in the human ABO-Rh blood typing system. Three independent donors of each blood type, same donor reproducibility, different conductivity buffers (0.52-9.1?mS/cm), and DC electric fields (17.1-68.5?V/cm) were tested to investigate separation dependencies. The data analysis was conducted from image intensity profiles across inlet and outlet channels in the device. Individual channel fractions suggest that the dielectrophoretic force experienced by the cells is dependent on erythrocyte antigen expression. Two different statistical analysis methods were conducted to determine how distinguishable a single blood type was from the others. Results indicate that channel fraction distributions differ by ABO-Rh blood types suggesting that antigens present on the erythrocyte membrane polarize differently in DC-iDEP fields. Under optimized conductivity and field conditions, certain blind blood samples could be sorted with low misclassification rates.     

Wirelessly powered microfluidic dielectrophoresis devices using printable RF circuits

We report the first microfluidic device integrated with a printed RF circuit so the device can be wirelessly powered by a commercially available RFID reader. For conventional dielectrophoresis devices, electrical wires are needed to connect the electric components on the microchip to external equipment such as power supplies, amplifiers, function generators, etc. Such a procedure is unfamiliar to most clinicians and pathologists who are used to working with a microscope for examination of samples on microscope slides. The wirelessly powered device reported here eliminates the entire need for wire attachments and external instruments so the operators can use the device in essentially the same manner as they do with microscope slides. The integrated circuit can be fabricated on a flexible plastic substrate at very low cost using a roll-to-roll printing method. Electrical power at 13.56 MHz transmitted by a radio-frequency identification (RFID) reader is inductively coupled to the printed RFIC and converted into 10 V DC (direct current) output, which provides sufficient power to drive a microfluidic device to manipulate biological particles such as beads and proteins via the DC dielectrophoresis (DC-DEP) effect. To our best knowledge, this is the first wirelessly powered microfluidic dielectrophoresis device. Although the work is preliminary, the device concept, the architecture, and the core technology are expected to stimulate many efforts in the future and transform the technology to a wide range of clinical and point-of-care applications.     

Live cell imaging analysis of the epigenetic regulation of the human endothelial cell migration at single-cell resolution

Epigenetic regulation plays an important role in cell migration. Although many methods have been developed to measure the motility of mammalian cells, accurate quantitative assessments of the migration speed of individual cells remain a major challenge. It is difficult for conventional scratch assays to differentiate proliferation from migration during the so-called wound-healing processes because of the long experimental time required. In addition, it is also challenging to create identical conditions for evaluating cell migration by conventional methods. We developed a microfluidic device with precisely created blanks allowing for robust and reproducible cell migration inside accurately-controlled microenvironments to study the regulatory effect of the epigenetic regulator histone deacetylase 7 (HDAC7) on cell migration. Through analyzing time-lapse imaging of the cells migrating into individual blank regions, we can measure the migration speed parameter for human primary cells within a few hours, eliminating the confounding effect of cell proliferation. We also developed an automatic image analysis and a numeric model-based data fitting to set up an integrated cell migration analysis system at single-cell resolution. Using this system, we measured the motility of primary human umbilical vein endothelial cells (HUVECs) and the migration speed reduction due to the silencing of HDAC7 and various other genes. We showed that the migration behaviour of these human primary cells are clearly regulated by epigenetic mechanisms, demonstrating the great potential of this accurate and robust assay in the fields of quantitatively migration studies and high-throughput screening.     

A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis

We have developed a prototype three-channel microfluidic chip that is capable of generating a linear concentration gradient within a microfluidic channel and is useful in the study of bacterial chemotaxis. The linear chemical gradient is established by diffusing a chemical through a porous membrane located in the side wall of the channel and can be established without through-flow in the channel where cells reside. As a result, movement of the cells in the center channel is caused solely by the cells chemotactic response and not by variations in fluid flow. The advantages of this microfluidic chemical linear gradient generator are (i) its ability to produce a static chemical gradient, (ii) its rapid implementation, and (iii) its potential for highly parallel sample processing. Using this device, wildtype Escherichia coli strain RP437 was observed to move towards an attractant (e.g., l-asparate) and away from a repellent (e.g., glycerol) while derivatives of RP437 that were incapable of motility or chemotaxis showed no bias of the bacteria's distribution. Additionally, the degree of chemotaxis could be easily quantified using this assay in conjunction with fluorescence imaging techniques, allowing for estimation of the chemotactic partition coefficient (CPC) and the chemotactic migration coefficient (CMC). Finally, using this approach we demonstrate that E. coli deficient in autoinducer-2-mediated quorum sensing respond to the chemoattractant l-aspartate in a manner that is indistinguishable from wildtype cells suggesting that chemotaxis is insulated from this mode of cell-cell communication.     

Toward low-voltage dielectrophoresis-based microfluidic systems: A review

Dielectrophoretically driven microfluidic devices have demonstrated great applicability in biomedical engineering, diagnostic medicine, and biological research. One of the potential fields of application for this technology is in point-of-care (POC) devices, ideally allowing for portable, fully integrated, easy to use, low-cost diagnostic platforms. Two main approaches exist to induce dielectrophoresis (DEP) on suspended particles, that is, electrode-based DEP and insulator-based DEP, each featuring different advantages and disadvantages. However, a shared concern lies in the input voltage used to generate the electric field necessary for DEP to take place. Therefore, input voltage can determine portability of a microfluidic device. This review outlines the recent advances in reducing stimulation voltage requirements in DEP-driven microfluidics.