Three‐dimensional microfluidic chip with twin‐layer herringbone structure for high efficient tumor cell capture and release via antibody‐conjugated magnetic microbeads

Harvesting rare circulating tumor cells (CTCs) from human blood is distinctly substantial to monitor tumor stage and evaluate therapeutic efficacy. As a proof‐of‐concept study, a microfluidic chip with twin‐layer herringbone grooves was developed to isolate and recover tumor cells with high efficiency based on the immunoreaction between cells and antibody‐conjugated microbeads (MBs) under local magnetic field. Functional MBs were initially localized onto the internal channel wall through the magnetic guidance. Then, infused tumor cells were deviated into the herringbone groove via passive microvortex and were further trapped through an irreversible interaction with MBs. Upon the removal of magnet, the captured cells and residual MBs were released from the channel and collected for further analysis in cell adhesion and proliferation in vitro. Capture efficiency of tumor cells reached up to ∼90% and limit of detection was down to 50 cells per mL based on this approach. Furthermore, recovery rate of tumor cells was as high as ∼94%, and potencies of cell attachment and proliferation was well maintained in retrieved cells. Hence, the present technique has a great potential for the isolation, quantitation and recovery of CTCs for cancer theranostic guidance and biomolecular analysis.     

Bacteria and cancer cell pearl chain under dielectrophoresis

In this work, we aim to observe and study the physics of bacteria and cancer cells pearl chain formation under dielectrophoresis (DEP). Experimentally, we visualized the formation of Bacillus subtilis bacterial pearl chain and human breast cancer cell (MCF-7) chain under positive and negative dielectrophoretic force, respectively. Through a simple simulation with creeping flow, AC/DC electric fields, and particle tracing modules in COMSOL, we examined the mechanism by which bacteria self-organize into a pearl chain across the gap between two electrodes via DEP. Our simulation results reveal that the region of greatest positive DEP force shifts from the electrode edge to the leading edge of the pearl chain, thus guiding the trajectories of free-flowing particles toward the leading edge via positive DEP. Our findings additionally highlight the mechanism why the free-flowing particles are more likely to join the existing pearl chain rather than starting a new pearl chain. This phenomenon is primarily due to the increase in magnitude of electric field gradient, and hence DEP force exerted, with the shortening gap between the pearl chain leading edge and the adjacent electrode. The findings shed light on the observed behavior of preferential pearl chain formation across electrode gaps.     

On the design of deterministic dielectrophoresis for continuous separation of circulating tumor cells from peripheral blood cells

Detection and analysis of circulating tumor cells (CTCs) have emerged as a promising way to diagnose cancer, study its cellular mechanism, and test or develop potential treatments. However, the rarity of CTCs among peripheral blood cells is a big challenge toward CTC detection. In addition, in cases where there is similar size range between certain types of CTCs (e.g. breast cancer cells) and white blood cells (WBCs), high‐resolution techniques are needed. In the present work, we propose a deterministic dielectrophoresis (DEP) method that combines the concept of deterministic lateral displacement (DLD) and insulator‐based dielectrophoresis (iDEP) techniques that rely on physical markers such as size and dielectric properties to differentiate different type of cells. The proposed deterministic DEP technology takes advantage of frequency‐controlled AC electric field for continuous separation of CTCs from peripheral blood cells. Utilizing numerical modeling, different aspects of coupled DLD‐DEP design such as the required applied voltages, velocities, and geometrical parameters of DLD arrays of microposts are investigated. Regarding the inevitable difference and uncertainty ranges for the reported crossover frequencies of cells, a comprehensive analysis is conducted on applied electric field frequency as design's determinant factor. Deterministic DEP design provides continuous sorting of CTCs from WBCs even with similar size and has the future potential for high throughput and efficiency.     

Scaling law analysis of electrohydrodynamics and dielectrophoresis for isomotive dielectrophoresis microfluidic devices

Isomotive dielectrophoresis (isoDEP) is a unique DEP geometrical configuration where the gradient of the field-squared () is constant. IsoDEP analyzes polarizable particles based on their magnitude and direction of translation. Particle translation is a function of the polarizability of both the particles and suspending medium, the particles’ size and shape, and the frequency of the electric field. However, other electrokinetics act on the particles simultaneously, including electrothermal hydrodynamics. Hence, to maximize the DEP force relative to over electrokinetic forces, design parameters such as microchannel geometry, fabrication materials, and applied electric field must be properly tuned. In this work, scaling law analyses were developed to derive design rules, relative to particle diameter, to reduce unwanted electrothermal hydrodynamics relative to DEP-induced particle translation. For a particle suspended in 10 mS/m media, if the channel width and height are below ten particle diameters, the electrothermal-driven flow is reduced by ∼500 times compared to a channel that is 250 particles diameters in width and height. Replacing glass with silicon as the device's underlying substrate for an insulative-based isoDEP reduces the electrothermal induced flow approximately 20 times less.     

Experimental study of dielectrophoresis and liquid dielectrophoresis mechanisms for particle capture in a droplet

This work presents a microfluidic system that can transport, concentrate, and capture particles in a controllable droplet. Dielectrophoresis (DEP), a phenomenon in which a force is exerted on a dielectric particle when it is subjected to a non-uniform electric field, is used to manipulate particles. Liquid dielectrophoresis (LDEP), a phenomenon in which a liquid moves toward regions of high electric field strength under a non-uniform electric field, is used to manipulate the fluid. In this study, a mechanism of droplet creation presented in a previous work that uses DEP and LDEP is improved. A driving electrode with a DEP gap is used to prevent beads from getting stuck at the interface between air and liquid, which is actuated with an AC signal of 200 V(pp) at a frequency of 100 kHz. DEP theory is used to calculate the DEP force in the liquid, and LDEP theory is used to analyze the influence of the DEP gap. The increment of the actuation voltage due to the electrode with a DEP gap is calculated. A set of microwell electrodes is used to capture a bead using DEP force, which is actuated with an AC signal of 20 V(pp) at a frequency of 5 MHz. A simulation is carried out to investigate the dimensions of the DEP gap and microwell electrodes. Experiments are performed to demonstrate the creation of a 100-nL droplet and the capture of individual 10-μm polystyrene latex beads in the droplet.     

Novel microfluidic device for the continuous separation of cancer cells using dielectrophoresis

We describe the design, microfabrication, and testing of a microfluidic device for the separation of cancer cells based on dielectrophoresis. Cancer cells, specifically green fluorescent protein‐labeled MDA‐MB‐231, are successfully separated from a heterogeneous mixture of the same and normal blood cells. MDA‐MB‐231 cancer cells are separated with an accuracy that enables precise detection and counting of circulating tumor cells present among normal blood cells. The separation is performed using a set of planar interdigitated transducer electrodes that are deposited on the surface of a glass wafer and slightly protrude into the separation microchannel at one side. The device includes two parts, namely, a glass wafer and polydimethylsiloxane element. The device is fabricated using standard microfabrication techniques. All experiments are conducted with low conductivity sucrose‐dextrose isotonic medium. The variation in response between MDA‐MB‐231 cancer cells and normal cells to a certain band of alternating‐current frequencies is used for continuous separation of cells. The fabrication of the microfluidic device, preparation of cells and medium, and flow conditions are detailed. The proposed microdevice can be used to detect and separate malignant cells from heterogeneous mixture of cells for the purpose of early screening for cancer.     

Accumulation and trapping of hepatitis A virus particles by electrohydrodynamic flow and dielectrophoresis

Hepatitis A virus particles (d = 27 nm) were successfully accumulated and trapped in a microfluidic system by means of a combination of electrohydrodynamic flow and dielectrophoretic forces. Electric fields were generated in a field cage consisting of eight microelectrodes. In addition, high medium conductance (0.3 S/m) resulted in sufficient Joule heating and the corresponding spatial variation of temperature, density, and permittivity to induce electrohydrodynamic flow in the vicinity of the field cage. Flow vortices transport particles toward the center of the field cage, where dielectrophoretic forces cause permanent entrapment and particle aggregation. Spatial distribution of temperature, density, and permittivity as well as resulting flow patterns were modeled numerically and are in good agreement with experimental results. This accumulation scheme might be applicable to sample concentration enhancement in biosensor applications.     

Microdevices for manipulation and accumulation of micro- and nanoparticles by dielectrophoresis

Microfluidic devices with three-dimensional (3-D) arrays of microelectrodes embedded in microchannels have been developed to study dielectrophoretic forces acting on synthetic micro- and nanoparticles. In particular, so-called deflector structures were used to separate particles according to their size and to enable accumulation of a fraction of interest into a small sample volume for further analysis. Particle velocity within the microchannels was measured by video microscopy and the hydrodynamic friction forces exerted on deflected particles were determined according to Stokes law. These results lead to an absolute measure of the dielectrophoretic forces and allowed for a quantitative test of the underlying theory. In summary, the influence of channel height, particle size, buffer composition, electric field, strength and frequency on the dielectrophoretic force and the effectiveness of dielectrophoretic deflection structures were determined. For this purpose, microfluidic devices have been developed comprising pairs of electrodes extending into fluid channels on both top and bottom side of the microfluidic channels. Electrodes were aligned under angles varying from 0 to 75 degrees with respect to the direction of flow. Devices with channel height varying between 5 and 50 microm were manufactured. Fabrication involved a dedicated bonding technology using a mask aligner and UV-curing adhesive. Particles with radius ranging from 250 nm to 12 microm were injected into the channels using aqueous buffer solutions.     

DNA fragment‐assisted microfluidic chip for capture and release of circulating tumor cells

Circulating tumor cells are specifically referred as cells that detached from the primary tumor and are present in the bloodstream. They could be isolated from blood and used as representative biomarker for predicting cancer prognoses. Here, we developed a microfluidic chip with multiple curved channels, in which DNA fragments and antibody‐based enrichment are exploited to capture circulating tumor cells in blood sample. By introducing DNA fragments as long tentacles, the active antibody could be extended into the microchannel stereoscopically, which could greatly increase the chances of adhesion in a multidirectional way and improve the capture efficacy. Several pivotal factors for cell capturing were optimized to the best state. Compared to conventional chips for planar capturing, the capture efficiency of MCF‐7 cells was greatly increased from 37.17 to 85.10%. For the detection of MCF‐7‐containing artificial blood sample detection, the capture efficiency of tumor cells was about 74.19 ± 2.13%, which was obviously better than the result of flow cytometry (29.67 ± 4.02%). Captured cells were easily released from the surface of microfluidic chip with high cell viability, which could be investigated for the molecular analysis in the field of tumor diagnosis.     

A single-view field filter device for rare tumor cell filtration and enumeration

In this work, we demonstrate a single-view field filter (SVFF) device for the efficient filtration and enumeration of rare tumor cells in the blood. In our device, the track-etched membrane is integrated within a low-cost polymer-film microfluidic chip, and multiplex microfiltration chambers are designed. Our device permits the performing of multiple sample tests on a single membrane and the dynamical observation of the entire filtration process in a single field of view. To characterize the device performance, our device is first tested using tumor cells, and three different cell behaviors are observed during the filtration process. Finally, we successfully apply our device for the separation of rare tumor cells from the lysed blood samples at various flow rates. The recovery rates of 93.3, 87.6, and 84.1% can be respectively achieved at the throughputs of 50, 100, and 150 μL/min. Our single-view field filter (SVFF) device offers the advantages of label-free filtration, efficient enumeration, easy integration, and low cost, and holds the potential to be used as an efficient tool for the filtration and enumeration of rare cells.     

Hydrodynamic gating valve for microfluidic fluorescence-activated cell sorting

Microfluidic cell sorter allows efficient separation of small number of cells, which is beneficial in handling cells, especially primary cells that cannot be expanded to large populations. Here, we demonstrate a microfluidic fluorescence-activated cell sorter (μFACS) with a novel sorting mechanism, in which automatic on-chip sorting is realized by turning on/off the hydrodynamic gating valve when a fluorescent target is detected. Formation of the hydrodynamic gating valve was investigated by both numerical simulation and flow visualization experiment. Separation of fluorescent polystyrene beads was then conducted to evaluate this sorting mechanism and to optimize the separation conditions. Isolation of fluorescent HeLa-DsRed cells was further demonstrated with high purity and recovery rate. Viability of the sorted cells was also examined, suggesting a survival rate of more than 90%. We expect this sorting approach to find widespread applications in bioanalysis.     

Numerical study of dielectrophoresis-modified inertial migration for overlapping sized cell separation

Circulating tumor cells (CTCs) have been proven to have significant prognostic, diagnostic, and clinical values in early-stage cancer detection and treatment. The efficient separation of CTCs from peripheral blood can ensure intact and viable CTCs and can, thus, give proper genetic characterization and drug innovation. In this study, continuous and high-throughput separation of MDA-231 CTCs from overlapping sized white blood cells (WBCs) is achieved by modifying inertial cell focusing with dielectrophoresis (DEP) in a single-stage microfluidic platform by numeric simulation. The DEP is enabled by embedding interdigitated electrodes with alternating field control on a serpentine microchannel to avoid creating two-stage separation. Rather than using the electrokinetic migration of cells which slows down the throughput, the system leverages the inertial microfluidic flow to achieve high-speed continuous separation. The cell migration and cell positioning characteristics are quantified through coupled physics analyses to evaluate the effects of the applied voltages and Reynolds numbers (Re) on the separation performance. The results indicate that the introduction of DEP successfully migrates WBCs away from CTCs and that separation of MDA-231 CTCs from similar sized WBCs at a high Re of 100 can be achieved with a low voltage of magnitude 4 ×10⁶ V/m. Additionally, the viability of MDA-231 CTCs is expected to be sustained after separation due to the short-term DEP exposure. The developed technique could be exploited to design active microchips for high-throughput separation of mixed cell beads despite their significant size overlap, using DEP-modified inertial focusing controlled simply by adjusting the applied external field.     

Bacteria capture, concentration and detection by alternating current dielectrophoresis and self-assembly of dispersed single-wall carbon nanotubes

The high polarizability and dielectrophoretic mobility of single-walled carbon nanotubes (SWNT) are utilized to capture and detect low numbers of bacteria and submicron particles in milliliter-sized samples. Concentrated SWNT solutions are mixed with the sample and a high-frequency (>100 kHz) alternating current (AC) field is applied by a microelectrode array to enhance bulk absorption of the particles (bacteria and nanoparticle substitutes) by the SWNTs via dipole-dipole interaction. The same AC field then drives the SWNT-bacteria aggregates to the microelectrode array by positive-AC dielectrophoresis (DEP), with enhanced and reversed bacteria DEP mobility due to the attached SWNTs. Since the field frequency exceeds the inverse RC time of the electrode double layer, the AC field penetrates deeply into the bulk and across the electrode gap. Consequently, the SWNTs and absorbed bacteria assemble rapidly (<5 min) into conducting linear aggregates between the electrodes. Measured AC impedance spectra by the same trapping electrodes and fields show a detection threshold of 10(4) bacteria/mL with this pathogen trapping and concentration technique.     

High-resolution analyses of cell fusion dynamics in a biochip

In this study, we analyzed the electrofusion of two cells in a biochip that has been developed to perform the capture by dielectrophoresis and the electrofusion of pairs of cells. The good transparency of the microsystem allowed analyzing the details of the fusion events. By staining one of the cells, the mixing of the two cytosols could be observed during the electrofusion experiment. We show for the first time the rapidity of the mixing of the two cytosols: less than 5 s under our experimental conditions. By comparing these experimental results to a numerical simulation, we found that the rate of this phenomenon is compatible with a diffusion-only mechanism, showing that during the fusion, the two cell membranes in contact are affected by very rapid structural changes and do not limit the exchange of the cytosols between the two cells. A point of interest is the use of dielectric structures to concentrate the electric field and of positive dielectrophoresis to capture cells in the area where the electric field is more intense. This technique allows the increase of the cell-to-cell contact and limits cell cytosol leakages during the fusion process.     

Detection of non‐small cell lung cancer cells based on microfluidic polarization microscopic image analysis

In early diagnosis of lung cancer, a polarization microscopy is a powerful tool to obtain the optical information of biological tissues. In this paper, a new microfluidic polarization imaging and analysis method was proposed for the detection and classification of cancer‐associated fibroblasts and the two kinds of non‐small cell lung cancer cells, A549 and H322. A polarizing microscopy system was constructed based on a commercial microscope to obtain 3*3 Mueller matrix of cells. Based on the Muller matrix decomposition algorithm and analysis in spatial domain and frequency domain, appropriate classification parameters were selected for the characterization of different polarization characteristics of cells. Finally, the logistic regression models based on machine learning were applied to determine optimal feature parameters and classify cells. This method integrated the morphological information of the cells, and the polarization characteristics of the cells in different polarization states. It is for the first time that the polarization microscopic image analysis method has been applied to the detection and classification of non‐small cell lung cancer cells. The results show that the presented microfluidic polarization microscopic image analysis method could classify cells effectively. Compared with the Muller matrix measurement and calculation methods, the method proposed in this paper was greatly simplified in both the acquisition of polarized images and the analysis and processing of polarized images.     

Spatially selective reagent delivery into cancer cells using a two-layer microfluidic culture system

In this work, we demonstrate a two-layer microfluidic system capable of spatially selective delivery of drugs and other reagents under low shear stress. Loading occurs by hydrodynamically focusing a reagent stream over a particular region of the cell culture. The system consisted of a cell culture chamber and fluid flow channel, which were located in different layers to reduce shear stress on cells. Cells in the center of the culture chamber were exposed to parallel streams of laminar flow, which allowed fast changes to be made to the cellular environment. The shear force was reduced to 2.7 dyn cm⁻² in the two-layer device (vs. 6.0 dyn cm⁻² in a one-layer device). Cells in the side of the culture chamber were exposed to the side streams of buffer; the shear force was further reduced to a greater extent since the sides of the culture chamber were separated from the main fluid path. The channel shape and flow rate of the multiple streams were optimized for spatially controlled reagent delivery. The boundaries between streams were well controlled at a flow rate of 0.1 mL h⁻¹, which was optimized for all streams. We demonstrated multi-reagent delivery to different regions of the same culture well, as well as selective treatment of cancer cells with a built in control group in the same well. In the case of apoptosis induction using staurosporine, 10% of cells remained viable after 24 h of exposure. Cells in the same chamber, but not exposed to staurosporine, had a viability of 90%. This chip allows dynamic observation of cellular behavior immediately after drug delivery, as well as long-term drug treatment with the benefit of large cell numbers, device simplicity, and low shear stress.     

In vitro dielectrophoresis of HEK cell migration for stimulating chronic wound epithelialization

This article describes a dielectrophoresis (DEP)-based simulation and experimental study of human epidermal keratinocyte (HEK) cells for wounded skin cell migration toward rapid epithelialization. MyDEP is a standalone software designed specifically to study dielectric particles and cell response to an alternating current (AC) electric field. This method demonstrated that negative dielectrophoresis (N_DEP) occurs in HEK cells at a wide frequency range in highly conductive medium. The finite element method was used to characterize particle trajectory based on DEP and drag force. The performance of the system was assessed using HEK cells in a highly conductive EpiLife suspending medium. The DEP experiment was performed by applying sinusoidal wave AC potential at the peak-to-peak voltage of 10 V in a tapered aluminum microelectrode array from 100 kHz to 1 MHz. We experimentally observed the occurrence of NDEP, which attracted HEK cells toward the local electric field minima in the region of interest. The DIPP-MotionV software was used to track cell migration in the prerecorded video via an automatic marker and estimate the average speed and acceleration of the cells. The results showed that HEK cell migration was accomplished approximately at 6.43 μm/s at 100 kHz with 10 V, and F_DEP caused the cells to migrate and align at the target position, which resulted in faster wound closures because of the application of an electric field frequency to HEK cells in random locations.     

Label-free enrichment of MCF7 breast cancer cells from leukocytes using continuous flow dielectrophoresis

Circulating tumor cells (CTCs) present in the bloodstream are strongly linked to the invasive behavior of cancer; therefore, their detection holds great significance for monitoring disease progression. Currently available CTC isolation tools are often based on tumor-specific antigen or cell size approaches. However, these techniques are limited due to the lack of a unique and universal marker for CTCs, and the overlapping size between CTCs and regular blood cells. Dielectrophoresis (DEP), governed by the intrinsic dielectric properties of the particles, is a promising marker-free, accurate, fast, and low-cost technique that enables the isolation of CTCs from blood cells. This study presents a continuous flow, antibody-free DEP-based microfluidic device to concentrate MCF7 breast cancer cells, a well-established CTC model, in the presence of leukocytes extracted from human blood samples. The enrichment strategy was determined according to the DEP responses of the corresponding cells, obtained in our previously reported DEP spectrum study. It was based on the positive-DEP integrated with hydrodynamic focusing under continuous flow. In the proposed device, the parylene microchannel with two inlets and outlets was built on top of rectangular and equally spaced isolated planar electrodes rotated certain degree relative to the main flow (13°). The recovery of MCF7 cells mixed with leukocytes was 74%–98% at a frequency of 1 MHz and a magnitude of 10–12 V_pp. Overall, the results revealed that the presented system successfully concentrates MCF7 cancer cells from leukocytes, ultimately verifying our DEP spectrum study, in which the enrichment frequency and separation strategy of the microfluidic system were determined.     

Breast cancer cell obatoclax response characterization using passivated‐electrode insulator‐based dielectrophoresis

Inherent electrical properties of cells can be beneficial to characterize different cell lines and their response to experimental drugs. This paper presents a novel method to characterize the response of breast cancer cells to drug stimuli through use of off‐chip passivated‐electrode insulator‐based dielectrophoresis (OπDEP) and the application of AC electric fields. This work is the first to demonstrate the ability of OπDEP to differentiate between two closely related breast cancer cell lines, LCC1 and LCC9 while assessing their drug sensitivity to an experimental anti‐cancer agent, Obatoclax. Although both cell lines are derivatives of estrogen‐responsive MCF‐7 breast cancer cells, growth of LCC1 is estrogen independent and anti‐estrogen responsive, while LCC9 is both estrogen‐independent and anti‐estrogen resistant. Under the same operating conditions, LCC1 and LCC9 had different DEP profiles. LCC1 cells had a trapping onset (crossover) frequency of 700 kHz and trapping efficiencies between 30–40%, while LCC9 cells had a lower crossover frequency (100 kHz) and showed higher trapping efficiencies of 40–60%. When exposed to the Obatoclax, both cell lines exhibited dose‐dependent shifts in DEP crossover frequency and trapping efficiency. Here, DEP results supplemented with cell morphology and proliferation assays help us to understand the response of these breast cancer cells to Obatoclax.