A handheld preconcentrator for the rapid collection of cancerous cells using dielectrophoresis generated by circular microelectrodes in stepping electric fields

The ability to concentrate biological cells, such as circulating tumor cells, circulating fetal cells, and stem cells, is an important issue in medical diagnostics and characterization. The present study develops a handheld device capable of effectively preconcentrating cancerous cells. Circular microelectrodes were designed to generate a stepping electric field by switching the electric field to an adjacent electrode pair by relays. Cancerous cells with a positive dielectrophoretic response are guided toward the center of the circular microelectrodes due to the region of high electric field between the adjacent electrodes being gradually decreased in the direction of the stepping electric field. Numerical simulations of the electric fields were performed to demonstrate the concept of the proposed design. The preconcentration of HeLa cells, which are a human cervical carcinoma cell line, was achieved in 160 s with an efficiency of around 76%, with an applied peak-to-peak voltage of 16 V at a frequency of 1 MHz.     

Combined AC electroosmosis and dielectrophoresis for controlled rotation of microparticles

Electrorotation is widely used for characterization of biological cells and materials using a rotating electric field. Generally, multiphase AC electric fields and quadrupolar electrode configuration are needed to create a rotating electric field for electrorotation. In this study, we demonstrate a simple method to rotate dielectrophoretically trapped microparticles using a stationary AC electric field. Coplanar interdigitated electrodes are used to create a linearly polarized nonuniform AC electric field. This nonuniform electric field is employed for dielectrophoretic trapping of microparticles as well as for generating electroosmotic flow in the vicinity of the electrodes resulting in rotation of microparticles in a microfluidic device. The rotation of barium titanate microparticles is observed in 2-propanol and methanol solvent at a frequency below 1 kHz. A particle rotation rate as high as 240 revolutions per minute is observed. It is demonstrated that precise manipulation (both rotation rate and equilibrium position) of the particles is possible by controlling the frequency of the applied electric field. At low frequency range, the equilibrium positions of the microparticles are observed between the electrode edge and electrode center. This method of particle manipulation is different from electrorotation as it uses induced AC electroosmosis instead of electric torque as in the case of electrorotation. Moreover, it has been shown that a microparticle can be rotated along its own axis without any translational motion.     

Characterization and separation of Cryptosporidium and Giardia cells using on-chip dielectrophoresis

Dielectrophoresis (DEP) has been shown to have significant potential for the characterization of cells and could become an efficient tool for rapid identification and assessment of microorganisms. The present work is focused on the trapping, characterization, and separation of two species of Cryptosporidium (C. parvum and C. muris) and Giardia lambia (G. lambia) using a microfluidic experimental setup. Cryptosporidium oocysts, which are 2-4 μm in size and nearly spherical in shape, are used for the preliminary stage of prototype development and testing. G. lambia cysts are 8–12 μm in size. In order to facilitate effective trapping, simulations were performed to study the effects of buffer conductivity and applied voltage on the flow and cell transport inside the DEP chip. Microscopic experiments were performed using the fabricated device and the real part of Clausius—Mossotti factor of the cells was estimated from critical voltages for particle trapping at the electrodes under steady fluid flow. The dielectric properties of the cell compartments (cytoplasm and membrane) were calculated based on a single shell model of the cells. The separation of C. muris and G. lambia is achieved successfully at a frequency of 10 MHz and a voltage of 3 Vpp (peak to peak voltage).     

Microfluidic separation of live and dead yeast cells using reservoir-based dielectrophoresis

Separating live and dead cells is critical to the diagnosis of early stage diseases and to the efficacy test of drug screening, etc. This work demonstrates a novel microfluidic approach to dielectrophoretic separation of yeast cells by viability. It exploits the cell dielectrophoresis that is induced by the inherent electric field gradient at the reservoir-microchannel junction to selectively trap dead yeast cells and continuously separate them from live ones right inside the reservoir. This approach is therefore termed reservoir-based dielectrophoresis (rDEP). It has unique advantages as compared to existing dielectrophoretic approaches such as the occupation of zero channel space and the elimination of any mechanical or electrical parts inside microchannels. Such an rDEP cell sorter can be readily integrated with other components into lab-on-a-chip devices for applications to biomedical diagnostics and therapeutics.     

Curvature-induced dielectrophoresis for continuous separation of particles by charge in spiral microchannels

The separation of particles from a heterogeneous mixture is critical in chemical and biological analyses. Many methods have been developed to separate particles in microfluidic devices. However, the majority of these separations have been limited to be size based and binary. We demonstrate herein a continuous dc electric field driven separation of carboxyl-coated and noncoated 10 μm polystyrene beads by charge in a double-spiral microchannel. This method exploits the inherent electric field gradients formed within the channel turns to manipulate particles by dielectrophoresis and is thus termed curvature-induced dielectrophoresis. The spiral microchannel is also demonstrated to continuously sort noncoated 5 μm beads, noncoated 10 μm beads, and carboxyl-coated 10 μm beads into different collecting wells by charge and size simultaneously. The observed particle separation processes in different situations are all predicted with reasonable agreements by a numerical model. This curvature-induced dielectrophoresis technique eliminates the in-channel microelectrodes and obstacles that are required in traditional electrode- and insulator-based dielectrophoresis devices. It may potentially be used to separate multiple particle targets by intrinsic properties for lab-on-a-chip applications.     

Isolation and enrichment of low abundant particles with insulator-based dielectrophoresis

Isolation and enrichment of low-abundant particles are essential steps in many bio-analytical and clinical applications. In this work, the capability of an insulator-based dielectrophoresis (iDEP) device for the detection and stable capture of low abundant polystyrene particles and yeast cells was evaluated. Binary and tertiary mixtures of particles and cells were tested, where the low-abundant particles had concentration ratios on the order of 1:10 000 000 compared to the other particles present in the mixture. The results demonstrated successful and stable capture and enrichment of rare particles and cells (trapping efficiencies over 99%), where particles remained trapped in a stable manner for up to 4 min. A device with four reservoirs was employed for the separation and enrichment of rare particles, where the particles of interest were first selectively concentrated and then effectively directed to a side port for future collection and analysis. The present study demonstrates that simple iDEP devices have appropriate screening capacity and can be used for handling samples containing rare particles; achieving both enrichment and isolation of low-abundant particles and cells.     

Antibody-independent isolation of circulating tumor cells by continuous-flow dielectrophoresis

Circulating tumor cells (CTCs) are prognostic markers for the recurrence of cancer and may carry molecular information relevant to cancer diagnosis. Dielectrophoresis (DEP) has been proposed as a molecular marker-independent approach for isolating CTCs from blood and has been shown to be broadly applicable to different types of cancers. However, existing batch-mode microfluidic DEP methods have been unable to process 10 ml clinical blood specimens rapidly enough. To achieve the required processing rates of 10⁶ nucleated cells/min, we describe a continuous flow microfluidic processing chamber into which the peripheral blood mononuclear cell fraction of a clinical specimen is slowly injected, deionized by diffusion, and then subjected to a balance of DEP, sedimentation and hydrodynamic lift forces. These forces cause tumor cells to be transported close to the floor of the chamber, while blood cells are carried about three cell diameters above them. The tumor cells are isolated by skimming them from the bottom of the chamber while the blood cells flow to waste. The principles, design, and modeling of the continuous-flow system are presented. To illustrate operation of the technology, we demonstrate the isolation of circulating colon tumor cells from clinical specimens and verify the tumor origin of these cells by molecular analysis.     

Separation by dielectrophoresis of dormant and nondormant bacterial cells of Mycobacterium smegmatis

The dielectrophoretic behavior of active, dead, and dormant Mycobacterium smegmatis bacterial cells was studied. It was found that the 72-h-old dormant cells had a much higher effective particle conductivity (812±10 μS cm⁻¹), almost double that of active cells (560±20 μS cm⁻¹), while that of dead (autoclaved) M. smegmatis cells was the highest (950±15 μS cm⁻¹) overall. It was also found that at 80 kHz, 900 μS cm⁻¹ dead cells were attracted at the edges of interdigitated castellated electrodes by positive dielectrophoresis, but dormant cells were not. Similarly, at 120 kHz, 2 μS cm⁻¹ active cells were attracted and dormant cells were not. Using these findings a dielectrophoresis-based microfluidic separation system was developed in which dead and active cells were collected from a given cell suspension, while dormant cells were eluted.     

Concurrent shear stress and chemical stimulation of mechano-sensitive cells by discontinuous dielectrophoresis

Microfluidic platforms enable a variety of physical or chemical stimulation of single or multiple cells to be examined and monitored in real-time. To date, intracellular calcium signalling research is, however, predominantly focused on observing the response of cells to a single mode of stimulation; consequently, the sensitising/desensitising of cell responses under concurrent stimuli is not well studied. In this paper, we provide an extended Discontinuous Dielectrophoresis procedure to investigate the sensitising of chemical stimulation, over an extensive range of shear stress, up to 63 dyn/cm², which encompasses shear stresses experienced in the arterial and venus systems (10 to 60 dyn/cm²). Furthermore, the TRPV4-selective agonist GSK1016790A, a form of chemical stimulation, did not influence the ability of the cells'' to remain immobilised under high levels of shear stress; thus, enabling us to investigate shear stress stimulation on agonism. Our experiments revealed that shear stress sensitises GSK1016790A-evoked intracellular calcium signalling of cells in a shear-stimulus dependent manner, as observed through a reduction in the cellular response time and an increase in the pharmacological efficacy. Consequently, suggesting that the role of TRPV4 may be underestimated in endothelial cells—which experience high levels of shear stress. This study highlights the importance of conducting studies at high levels of shear stress. Additionally, our approach will be valuable for examining the effect of high levels of shear on different cell types under different conditions, as presented here for agonist activation.     

Fabrication and characterization of nanomaterial-based sensors using dielectrophoresis

Dielectrophoresis (DEP) is an electrokinetic motion of dielectrically polarized materials in nonuniform electric fields. DEP has been successfully applied to manipulation of nanomaterials including carbon nanotubes (CNTs), metallic nanoparticles, and semiconducting nanowires. Under positive DEP force, which attracts nanomaterials toward the higher field region, nanomaterials are trapped in the electrode gap and automatically establish good electrical connections between them and the external measuring circuit. This feature allows us a fast, simple, and low-cost fabrication of nanomaterial-based sensors based on a bottom-up approach. This paper first presents a theoretical background of DEP phenomena and then reviews recent works of the present author, which were aimed to develop nanomaterial-based sensors, such as a CNT gas sensor and a ZnO nanowire photosensor, using DEP fabrication technique. It is also demonstrated that DEP technique enables self-formation of interfaces between various nanomaterials, which can be also applicable as novel sensing transducers.     

Separation of platelets from other blood cells in continuous-flow by dielectrophoresis field-flow-fractionation

We present a microfluidic device capable of separating platelets from other blood cells in continuous flow using dielectrophoresis field-flow-fractionation. The use of hydrodynamic focusing in combination with the application of a dielectrophoretic force allows the separation of platelets from red blood cells due to their size difference. The theoretical cell trajectory has been calculated by numerical simulations of the electrical field and flow speed, and is in agreement with the experimental results. The proposed device uses the so-called "liquid electrodes" design and can be used with low applied voltages, as low as 10 V(pp). The obtained separation is very efficient, the device being able to achieve a very high purity of platelets of 98.8% with less than 2% cell loss. Its low-voltage operation makes it particularly suitable for point-of-care applications. It could further be used for the separation of other cell types based on their size difference, as well as in combination with other sorting techniques to separate multiple cell populations from each other.     

Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis

Ovarian cancer is the leading cause of death from gynecological malignancies in women. The primary challenge is the detection of the cancer at an early stage, since this drastically increases the survival rate. In this study we investigated the dielectrophoretic responses of progressive stages of mouse ovarian surface epithelial (MOSE) cells, as well as mouse fibroblast and macrophage cell lines, utilizing contactless dielectrophoresis (cDEP). cDEP is a relatively new cell manipulation technique that has addressed some of the challenges of conventional dielectrophoretic methods. To evaluate our microfluidic device performance, we computationally studied the effects of altering various geometrical parameters, such as the size and arrangement of insulating structures, on dielectrophoretic and drag forces. We found that the trapping voltage of MOSE cells increases as the cells progress from a non-tumorigenic, benign cell to a tumorigenic, malignant phenotype. Additionally, all MOSE cells display unique behavior compared to fibroblasts and macrophages, representing normal and inflammatory cells found in the peritoneal fluid. Based on these findings, we predict that cDEP can be utilized for isolation of ovarian cancer cells from peritoneal fluid as an early cancer detection tool.     

Differential electronic detector to monitor apoptosis using dielectrophoresis-induced translation of flowing cells (dielectrophoresis cytometry)

The instrument described here is an all-electronic dielectrophoresis (DEP) cytometer sensitive to changes in polarizability of single cells. The important novel feature of this work is the differential electrode array that allows independent detection and actuation of single cells within a short section ( ～ 300?μm) of the microfluidic channel. DEP actuation modifies the altitude of the cells flowing between two altitude detection sites in proportion to cell polarizability; changes in altitude smaller than 0.25 μm can be detected electronically. Analysis of individual experimental signatures allows us to make a simple connection between the Clausius-Mossotti factor (CMF) and the amount of vertical cell deflection during actuation. This results in an all-electronic, label-free differential detector that monitors changes in physiological properties of the living cells and can be fully automated and miniaturized in order to be used in various online and offline probes and point-of-care medical applications. High sensitivity of the DEP cytometer facilitates observations of delicate changes in cell polarization that occur at the onset of apoptosis. We illustrate the application of this concept on a population of Chinese hamster ovary (CHO) cells that were followed in their rapid transition from a healthy viable to an early apoptotic state. DEP cytometer viability estimates closely match an Annexin V assay (an early apoptosis marker) on the same population of cells.     

Microfluidic size separation of cells and particles using a swinging bucket centrifuge

Biomolecular separation is crucial for downstream analysis. Separation technique mainly relies on centrifugal sedimentation. However, minuscule sample volume separation and extraction is difficult with conventional centrifuge. Furthermore, conventional centrifuge requires density gradient centrifugation which is laborious and time-consuming. To overcome this challenge, we present a novel size-selective bioparticles separation microfluidic chip on a swinging bucket minifuge. Size separation is achieved using passive pressure driven centrifugal fluid flows coupled with centrifugal force acting on the particles within the microfluidic chip. By adopting centrifugal microfluidics on a swinging bucket rotor, we achieved over 95% efficiency in separating mixed 20 μm and 2 μm colloidal dispersions from its liquid medium. Furthermore, by manipulating the hydrodynamic resistance, we performed size separation of mixed microbeads, achieving size efficiency of up to 90%. To further validate our device utility, we loaded spiked whole blood with MCF-7 cells into our microfluidic device and subjected it to centrifugal force for a mere duration of 10 s, thereby achieving a separation efficiency of over 75%. Overall, our centrifugal microfluidic device enables extremely rapid and label-free enrichment of different sized cells and particles with high efficiency.     

Microfluidic platform for separation and extraction of plasma from whole blood using dielectrophoresis

Microfluidic based blood plasma extraction is a fundamental necessity that will facilitate many future lab-on-a-chip based point-of-care diagnostic systems. However, current approaches for providing this analyte are hampered by the requirement to provide external pumping or dilution of blood, which result in low effective yield, lower concentration of target constituents, and complicated functionality. This paper presents a capillary-driven, dielectrophoresis-enabled microfluidic system capable of separating and extracting cell-free plasma from small amounts of whole human blood. This process takes place directly on-chip, and without the requirement of dilution, thus eliminating the prerequisite of pre-processed blood samples and external liquid handling systems. The microfluidic chip takes advantage of a capillary pump for driving whole blood through the main channel and a cross flow filtration system for extracting plasma from whole blood. This filter is actively unblocked through negative dielectrophoresis forces, dramatically enhancing the volume of extracted plasma. Experiments using whole human blood yield volumes of around 180 nl of cell-free, undiluted plasma. We believe that implementation of various integrated biosensing techniques into this plasma extraction system could enable multiplexed detection of various biomarkers.     

Characterization of microfluidic shear-dependent epithelial cell adhesion molecule immunocapture and enrichment of pancreatic cancer cells from blood cells with dielectrophoresis

Current microfluidic techniques for isolating circulating tumor cells (CTCs) from cancer patient blood are limited by low capture purity, and dielectrophoresis (DEP) has the potential to complement existing immunocapture techniques to improve capture performance. We present a hybrid DEP and immunocapture Hele-Shaw flow cell to characterize DEP''s effects on immunocapture of pancreatic cancer cells (Capan-1, PANC-1, and BxPC-3) and peripheral blood mononuclear cells (PBMCs) with an anti-EpCAM (epithelial cell adhesion molecule) antibody. By carefully specifying the applied electric field frequency, we demonstrate that pancreatic cancer cells are attracted to immunocapture surfaces by positive DEP whereas PBMCs are repelled by negative DEP. Using an exponential capture model to interpret our capture data, we show that immunocapture performance is dependent on the applied DEP force sign and magnitude, cell surface EpCAM expression level, and shear stress experienced by cells flowing in the capture device. Our work suggests that DEP can not only repel contaminating blood cells but also enhance capture of cancer cell populations that are less likely to be captured by traditional immunocapture methods. This combination of DEP and immunocapture techniques to potentially increase CTC capture purity can facilitate subsequent biological analyses of captured CTCs and research on cancer metastasis and drug therapies.     

利用粒子间相互作用的量子系统的纯度及相干性补偿

研究了混态粒子的纯度补偿以及消相干过程的抵消问题,提出了利用辅助粒子及其与目标粒子的相互作用进行纯度及相干性补偿的方法。给出了2种不同情况——混态目标粒子的纯度补偿和消相干过程的抵消——辅助粒子的充要条件。并通过系统数值仿真实验验证了所设计方法的有效性。     

Separation of particles and macromolecules by phase partition

The separation of simple organic substances by distribution between two immiscible phases is a traditional technique in preparative and analytical chemistry. Latterly, the method has been extended to cover the increasingly important field of polymers—especially those of biological importance—and the micro constituents of cells. By appropriate choice of phases the method can be made highly selective, and it is particularly valuable for the treatment of labile substances that may be irreversibly damaged by conventional methods.     

AC-dielectrophoretic characterization and separation of submicron and micron particles using sidewall AgPDMS electrodes

The recent development of microfluidic “lab on a chip” devices requires the need to continuously separate submicron particles. Here, we present a PDMS microfluidic device with sidewall conducting PDMS (AgPDMS) composite electrodes capable of separating submicron particles in hydrodynamic flow. In particular, the device can service dual functions. First, the AgPDMS composite electrodes embedded in a sidewall of the device channel allow for performing AC-dielectrophoretic (DEP) characterization through direct microscopic observation of particle behavior. Characterization experiments are carried out for numerous parameters including particle size, medium conductivity, and AC field frequency to reveal important dielectrophoresis DEP information in terms of the crossover frequency and positive/negative DEP behavior under specific frequencies. Second, the device offers an advantage that sidewall AgPDMS composite electrodes can produce strong DEP effects throughout the entire channel height, and thus the robustness of the on-chip particle separation is demonstrated for continuous separation in a flowing mixture of 0.5 and 5 μm particles with 100% separation efficiency.     

Transport of particles and microorganisms in microfluidic channels using rectified ac electro-osmotic flow

A new method is demonstrated to transport particles, cells, and other microorganisms using rectified ac electro-osmotic flows in open microchannels. The rectified flow is obtained by synchronous zeta potential modulation with the driving potential in the microchannel. Experiments were conducted to transport both neutral, charged particles, and microorganisms of various sizes. A maximum speed of 50 μm∕s was obtained for 8 μm polystyrene beads, without any electrolysis, using a symmetrical square waveform driving electric field of 5 V∕mm at 10 Hz and a 360 V gate potential with its polarity synchronized with the driving potential (phase lag=0°).