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.     

Investigation of viscoelastic focusing of particles and cells in a zigzag microchannel

Microfluidic particle focusing has been a vital prerequisite step in sample preparation for downstream particle separation, counting, detection, or analysis, and has attracted broad applications in biomedical and chemical areas. Besides all the active and passive focusing methods in Newtonian fluids, particle focusing in viscoelastic fluids has been attracting increasing interest because of its advantages induced by intrinsic fluid property. However, to achieve a well-defined focusing position, there is a need to extend channel lengths when focusing micrometer-sized or sub-microsized particles, which would result in the size increase of the microfluidic devices. This work investigated the sheathless viscoelastic focusing of particles and cells in a zigzag microfluidic channel. Benefit from the zigzag structure of the channel, the channel length and the footprint of the device can be reduced without sacrificing the focusing performance. In this work, the viscoelastic focusing, including the focusing of 10 μm polystyrene particles, 5 μm polystyrene particles, 5 μm magnetic particles, white blood cells (WBCs), red blood cells (RBCs), and cancer cells, were all demonstrated. Moreover, magnetophoretic separation of magnetic and nonmagnetic particles after viscoelastic pre-focusing was shown. This focusing technique has the potential to be used in a range of biomedical applications.     

Continuous blood cell separation by hydrophoretic filtration

We propose a new hydrophoretic method for continuous blood cell separation using a microfluidic device composed of slanted obstacles and filtration obstacles. The slanted obstacles have a larger height and gap than the particles in order to focus them to a sidewall by hydrophoresis. In the successive structure, the height and gap of the filtration obstacles with a filtration pore are set between the diameters of small and large particles, which defines the critical separation diameter. Accordingly, the particles smaller than the criterion freely pass through the gap and keep their focused position. In contrast, the particles larger than the criterion collide against the filtration obstacle and move into the filtration pore. The microfluidic device was characterized with polystyrene beads with a minimum diameter difference of 7.3%. We completely separated polystyrene microbeads of 9 and 12 microm diameter with a separation resolution of approximately 6.2. This resolution is increased by 6.4-fold compared with our previous separation method based on hydrophoresis (S. Choi and J.-K. Park, Lab Chip, 2007, 7, 890, ref. 1). In the isolation of white blood cells (WBCs) from red blood cells (RBCs), the microfluidic device isolated WBCs with 210-fold enrichment within a short filtration time of approximately 0.3 s. These results show that the device can be useful for the binary separation of a wide range of biological particles by size. The hydrophoretic filtration as a sample preparation unit offers potential for a power-free cell sorter to be integrated into disposable lab-on-a-chip devices.     

Fractionation and characterization of starch granules using field-flow fractionation (FFF) and differential scanning calorimetry (DSC)

Starch is one of the main carbohydrates in food; it is formed by two polysaccharides: amylose and amylopectin. The granule size of starch varies with different botanical origins and ranges from less than 1 μm to more than 100 μm. Some physicochemical and functional properties vary with the size of the granule, which makes it of great interest to find an efficient and accurate size-based separation method. In this study, the full-feed depletion mode of split-flow thin cell fractionation (FFD-SF) was employed for a size-based fractionation of two types of starch granules (corn and potato) on a large scale. The fractionation efficiency (FE) of fraction-a for corn and potato granules was 98.4 and 99.4%, respectively. The FFD-SF fractions were analyzed using optical microscopy (OM) and gravitational field-flow fractionation (GrFFF). The respective size distribution results were in close agreement for the corn starch fractions, while they were slightly different for the potato starch fractions. The thermal properties of FFD-SF fractions were analyzed, and the results for the potato starch showed that the peak temperature of gelatinization (T_p) slightly decreases as the size of the granules increases. Additionally, the enthalpy of gelatinization (ΔH) increases when the granule size increases and shows negative correlation with the gelatinization range (ΔT).

     

Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach.     

Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation

Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ～10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.     

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.     

Simultaneous isolation and label‐free identification of bacteria using contactless dielectrophoresis and Raman spectroscopy

The traditional bacterial identification method of growing colonies on agar plates can take several days to weeks to complete depending on the growth rate of the bacteria. Successfully decreasing this analysis time requires cell isolation followed by identification. One way to decrease analysis time is by combining dielectrophoresis (DEP), a common technique used for cell sorting and isolation, and Raman spectroscopy for cell identification. DEP‐Raman devices have been used for bacterial analysis, however, these devices have a number of drawbacks including sample heating, cell‐to‐electrode proximity that limits throughput and separation efficiency, electrode fouling, or inability to address sample debris. Presented here is a contactless DEP‐Raman device to simultaneously isolate and identify particles from a mixed sample while avoiding common drawbacks associated with other DEP designs. Using the device, a mixed sample of bacteria and 3 μm polystyrene spheres were isolated from each other and a Raman spectrum of the trapped bacteria was acquired, indicating the potential for cDEP‐Raman devices to decrease the analysis time of bacteria.     

Dielectric spectroscopy of red blood cells in sickle cell disease

Hypoxia-induced polymerization of sickle hemoglobin and the related ion diffusion across cell membrane can lead to changes in cell dielectric properties, which can potentially serve as label-free, diagnostic biomarkers for sickle cell disease. This article presents a microfluidic-based approach with on-chip gas control for the impedance spectroscopy of suspended cells within the frequency range of 40 Hz to 110 MHz. A comprehensive bioimpedance of sickle cells under both normoxia and hypoxia is achieved rapidly (within ∼7 min) and is appropriated by small sample volumes (∼2.5 μL). Analysis of the sensing modeling is performed to obtain optimum conditions for dielectric spectroscopy of sickle cell suspensions and for extraction of single cell properties from the measured impedance spectra. The results of sickle cells show that upon hypoxia treatment, cell interior permittivity and conductivity increase, while cell membrane capacitance decreases. Moreover, the relative changes in cell dielectric parameters are found to be dependent on the sickle and fetal hemoglobin levels. In contrast, the changes in normal red blood cells between the hypoxia and normoxia states are unnoticeable. The results of sickle cells may serve as a reference to design dielectrophoresis-based cell sorting and electrodeformation testing devices that require cell dielectric characteristics as input parameters. The demonstrated method for dielectric characterization of single cells from the impedance spectroscopy of cell suspensions can be potentially applied to other cell types and under varied gas conditions.     

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.     

Dielectrophoretic separation of platelets from diluted whole blood in microfluidic channels

The dielectrophoresis (DEP) phenomenon is used to separate platelets directly from diluted whole blood in microfluidic channels. By exploiting the fact that platelets are the smallest cell type in blood, we utilize the DEP-activated cell sorter (DACS) device to perform size-based fractionation of blood samples and continuously enrich the platelets in a label-free manner. Cytometry analysis revealed that a single pass through the two-stage DACS device yields a high purity of platelets (approximately 95%) at a throughput of approximately 2.2 x 10(4) cells/second/microchannel with minimal platelet activation. This work demonstrates gentle and label-free dielectrophoretic separation of delicate cells from complex samples and such a separation approach may open a path toward continuous screening of blood products by integrated microfluidic devices.     

Sheath-assisted versus sheathless dielectrophoretic particle separation

Lab-on-chip devices are widely being used for binary and ternary cell/particle separation applications. Among the lab-on-chip methods, dielectrophoresis (DEP) is a cost-effective and label-free method, with great capabilities for size-based separation of cells and particles, which is mostly performed in sheath-assisted forms. However, the elimination of the sheath flows offers advantages such as ease of operation and higher sample throughput. In this work, we present a comparison of sheath-assisted and sheathless DEP separation of three sizes of microparticles using tilted electrodes. The sheath-assisted design was capable of separating the 5, 10, and 15 μm particles with a separation efficiency as high as 98.0% for 15 μm particles. By adding a DEP focusing region, a sheathless DEP separator was proposed, which offered higher throughputs (up to 10 times) at the cost of lowering the separation efficiency (a reduction up to 10.3% for 15 μm) compared to the sheath-assisted design. To enhance the separation efficiency, a combination of the DEP focusing accompanied by weak sheath flows from both sides was proposed. This design achieved the highest sample separation yield in the outlets (as high as 98.7% for 15 μm) with a sample throughput of more than 4.2 μL/min. This study provides insights into the choice of an appropriate platform for any application in which the yield, purity, throughput, and portability must be considered.     

Optimized hybrid dielectrophoretic microchip for separation of bioparticles

Point-of-care diagnostics requires a smart separation of particles and/or cells. In this work, the multiorifice fluid fractionation as a passive method and dielectrophoresis-based actuator as an active tool are combined to offer a new device for size-based particle separation. The main objective of the combination of these two well-established techniques is to improve the performance of the multiorifice fluid fractionation by taking advantage of dielectrophoresis-based actuator for separating particles. Initially, by using numerical simulations, the effect of using dielectrophoresis-based actuator in multiorifice fluid fractionation on the separation of particles was investigated, and the size of the device was optimized by 25% compared to a device without dielectrophoresis-based actuator. Also, adding dielectrophoresis-based actuator to multiorifice fluid fractionation can extend the range of flow rates needed for separation. In the absence of dielectrophoresis-based actuator, the separation took place only when the flow rate is 100 μL/min, in the presence of dielectrophoresis-based actuator (20 Vp-p), the separation happened in flow rates ranging from 70 to 120 μL/min.     

A passive microfluidic device for continuous microparticle enrichment

A passive microfluidic device is reported for continuous microparticle enrichment. The microparticle is enriched based on the inertial effect in a microchannel with contracting‐expanding structures on one side where microparticles/cells are subjected to the inertial lift force and the momentum‐change‐induced inertial force induced by highly curved streamlines. Under the combined effect of the two forces, yeast cells and microparticles of different sizes were continuously focused in the present device over a range of Reynolds numbers from 16.7 to 125. ～68% of the particle‐free liquid was separated from the sample at Re = 66.7, and ～18 μL particle‐free liquid was fast obtained within 10 s. Results also showed that the geometry of the contracting‐expanding structure significantly influenced the lateral migration of the particle. Structures with a large angle induced strong inertial effect and weak disturbance effect of vortex on the particle, both of which enhanced the microparticle enrichment in microchannel. With simple structure, small footprint (18 × 0.35 mm), easy operation and cell‐friendly property, the present device has great potential in biomedical applications, such as the enrichment of cells and the fast extraction of plasma from blood for disease diagnose and therapy.     

Mechanical property analysis of stored red blood cell using optical tweezers

The deformation of human red blood cells subjected to direct stretching by optical tweezers was analyzed. The maximum force exerted by optical tweezers on the cell via a polystyrene microbead 5 μm in diameter was 315 pN. Digital image correlation (DIC) method was introduced to calculate the force and the deformation of the cell for the first time. Force–extension relation curves of the biconcave cell were quantitatively assessed when erythrocytes were stored in Alsever's Solution for 2 days, 5 days, 7 days and 14 days respectively. Experiment results demonstrated that the deformability of red blood cells was impaired with the stored time.     

Electrochemical hematocrit determination in a direct current microfluidic device

Hematocrit (HCT) tests are widely performed to screen blood donors and to diagnose medical conditions. Current HCT test methods include conventional microhematocrit, Coulter counter, CuSO₄ specific gravity, and conductivity‐based point‐of‐care (POC) HCT devices, which can be either expensive, environmentally inadvisable, or complicated. In the present work, we introduce a new and simple microfluidic system for a POC HCT determination. HCT was determined by measuring current responses of blood under 100 V DC for 1 min in a microfluidic device containing a single microchannel with dimensions of 180 μm by 70 μm and 10 mm long. Current responses of red blood cell (RBC) suspensions in PBS or separately plasma at HCT concentrations of 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, and 70 vol% were measured to show feasibility of the microfluidic system for HCT determination. Key parameters affecting current responses included electrolysis bubbles and irreversible RBC adsorption; parameters were optimized via addition of nonionic surfactant Triton X‐100 into sample solution and carbonizing electrode surfaces. The linear trend line of current responses over a range of RBC concentrations were obtained in both PBS and plasma. This work suggested that a simple microfluidic device could be a promising platform for a new POC HCT device.     

Combined detection of AC‐electrokinetic effects: Experiments with three‐axial chicken red blood cells

Dielectrophoresis (DEP), electrorotation (ROT), and electro‐orientation were used for the dielectric spectroscopy of nucleated three‐axial chicken red blood cells (CRBCs). Because the different AC‐electrokinetic effects are not mutually independent, their DEP and ROT spectra were combined in ranges separated by the reorientation of the CRBCs in the inhomogeneous linear DEP and circular ROT fields. This behavior can be qualitatively described by a single‐shell ellipsoidal model. Whereas in linear fields, the maximum of the Clausius–Mossotti factor along the three axes determines the orientated axis, in circular fields, the minimum of the factor determines the axis perpendicularly orientated to the field plane. Quantitatively, it has not been possible to find a consistent parameter set for fitting the DEP and ROT spectra, as well as the reorientation frequencies. Our ellipsoidal CRBC standard model had semiaxes of a = 7.7 μm, b = 4.0 μm, and c = 1.85 μm, a relative permittivity of 35 to 45 and conductivity of 0.36 to 0.04 S/m for the cytoplasm, combined with a specific capacitance of 10 to 14 mF/m² and a conductivity of 3500 S/m² for the cell membrane. The fits in different external conductivity ranges between external conductivities of 0.015 and 1.0 S/m were improved when the membrane capacitance was changed between 4 to 25 mF/m² depending on the method used. A similar transition was reflected in the effective properties of a three‐shell spherical model containing an internal membranous sphere with the geometry of the CRBC nucleus. Our findings suggest that the simultaneous interpretation of various AC‐electrokinetic spectra is a step toward the dielectric fingerprinting of biological cells.     

Rapid electrophoretic monitoring of the anaesthetic ketamine and its metabolite norketamine in rat blood using a contactless conductivity detector to study the pharmacokinetics

A method of capillary electrophoresis with contactless conductivity detection has been developed for non‐enantioselective monitoring the anaesthetic ketamine and its main metabolite norketamine. The separation is performed in a 15 μm capillary with an overall length of 31.5 cm and length to detector of 18 cm; inner surface of the capillary is covered with a commercial coating solution to reduce the electroosmotic flow. In an optimised background electrolyte with composition 2 M acetic acid + 1% v/v coating solution under application of a high voltage of 30 kV, the migration time is 97.1 s for ketamine and 95.8 s for norketamine, with an electrophoretic resolution of 1.2. The attained detection limit was 83 ng/mL (0.3 μmol/L) for ketamine and 75 ng/mL (0.3 μmol/L) for norketamine; the number of theoretic plates for separation of an equimolar model mixture with a concentration of 2 μg/mL was 683 500 plates/m for ketamine and 695 400 plates/m for norketamine. Laboratory preparation of rat blood plasma is based on mixing 10 μL of plasma with 30 μL of acidified acetonitrile, followed by centrifugation. A pharmacokinetic study demonstrated an exponential decrease in the plasma concentration of ketamine after intravenous application and much slower kinetics for intraperitoneal application.     

Hydrodynamic lift of vesicles and red blood cells in flow — from Fåhræus & Lindqvist to microfluidic cell sorting

Hydrodynamic lift forces acting on cells and particles in fluid flow receive ongoing attention from medicine, mathematics, physics and engineering. The early findings of Fåhræus & Lindqvist on the viscosity change of blood with the diameter of capillaries motivated extensive studies both experimentally and theoretically to illuminate the underlying physics. We review this historical development that led to the discovery of the inertial and non-inertial lift forces and elucidate the origins of these forces that are still not entirely clear. Exploiting microfluidic techniques induced a tremendous amount of new insights especially into the more complex interactions between the flow field and deformable objects like vesicles or red blood cells. We trace the way from the investigation of single cell dynamics to the recent developments of microfluidic techniques for particle and cell sorting using hydrodynamic forces. Such continuous and label-free on-chip cell sorting devices promise to revolutionize medical analyses for personalized point-of-care diagnosis. We present the state-of-the-art of different hydrodynamic lift-based techniques and discuss their advantages and limitations.     

Parametric simulation of the back-surface field effect in the silicon solar cell

Recombination of minority carriers in the solar cell is a major contributing factor in the loss of quantum efficiency and cell power. While the surface recombination is dealt with by depositing a passivation layer of SiO₂ or SiN_x, the bulk recombination is minimized by use of nearly defect-free monocrystalline substrate. In addition, the back-surface field (BSF) effect has been very useful in aiding the separation of free electrons and holes in the bulk. In this study, the key BSF parameters and their effect on the performance of a typical p-type front-lit Si solar cell are investigated by use of Medici, a 2-dimensional device simulator. Of the parameters, the doping concentration of the BSF layer is found to be most significant. That is, for a p-type substrate of 1 × 10¹⁴ cm⁻³ acceptor concentration, the optimum doping concentration of the BSF layer is 1 × 10¹⁸ cm⁻³ or more, and the maximum cell power can be increased by 24%, i.e., 25.4 mW cm⁻² vs. 20.5 mW cm⁻², by using a BSF layer with optimum doping. With regards to the BSF layer thickness, the impact is less. That is, the maximum cell power is about 11% higher at 100 μm than at 5 μm, which translates to an increase of 1.2% μm⁻¹. In practice, therefore, it would be better to rely on the control of the doping concentration than the thickness in maximizing the BSF effect in real Si solar cells.