A discrete-particle model of blood dynamics in capillary vessels

We investigate the mechanism of aggregation of red blood cells (RBC) in capillary vessels. We use a discrete-particle model in 3D to model the flow of plasma and RBCs within a capillary tube. This model can accurately capture the scales from 0.001 to 100 microm, far below the scales that can be modeled numerically with classical computational fluid dynamics. The flexible viscoelastic red blood cells and the walls of the elastic vessel are made up of solid particles held together by elastic harmonic forces. The plasma is represented by a system of dissipative fluid particles. Modeling has been carried out using 1 to 3 million solid and fluid particles. We have modeled the flow of cells with vastly different shapes, such as normal and "sickle" cells. The two situations involving a straight capillary and a pipe with a choking point have been considered. The cells can coagulate in spite of the absence of adhesive forces in the model. We conclude that aggregation of red blood cells in capillary vessels can be stimulated by depletion forces and hydrodynamic interactions. The cluster of "sickle" cells formed in the choking point of the capillary efficiently decelerates the flow, while normal cells can pass through. These qualitative results from our first numerical results accord well with the laboratory findings.     

Biophysical measurement of red blood cells by laboratory on print circuit board chip

Microfluidic impedance pulse sensor has emerged as an easily handled and low‐cost platform in the electrical analysis of biological cells. In the conventional method, impedance sensor demanded expensive patterning metal electrodes on the substrate, which are directly in touch with electrolytes in order to measure the microfluidic channel impedance change. In this article, a cost‐effective microfluidic impedance sensor built upon a dielectric film coated printed circuit board is introduced. Impedance electrodes are protected by a dielectric film layer from electrochemical erosion between electrodes and electrolyte. Human red blood cells from adult and neonatal were utilized to demonstrate the feasibility of the proposed device in the electroanalysis of biological cells.     

An efficient cell separation system using 3D-asymmetric microelectrodes

An efficient 3D-asymmetric microelectrode system for high-throughput was designed and fabricated to enhance sorting sensitivities to the dielectric properties-size, morphology, conductivity, and permittivity-of living cells. The principle of the present system is based on the use of the relative strengths of negative dielectrophoretic and drag forces, as in a conventional 3D-microelectrode system. Whereas the typical 3D-microelectrode system has a constant electric field magnitude due to the constant width of the microelectrodes and a fixed gap between face-to-face microelectrodes, the present 3D-asymmetric microelectrode system has electric fields of continuously varying magnitudes along the transverse direction of a channel owing to the changing widths of the electrodes in the half-circular shaped cross section of the microchannel. Thus, varying dielectric forces are generated, leading to increased sorting sensitivity through differentially induced forces to definitely distinct cell types. Numerical analysis verified the improved sensitivity of the present system for sorting living cells. The feasibility of using the newly fabricated system under experimental conditions was tested by demonstrating that a mixed population of mouse P19 embryonic carcinoma (EC) and red blood cells (RBCs) was effectively sorted to different wells depending on their respective relative physical properties.     

Investigation of pumping mechanism for non‐Newtonian blood flow with AC electrothermal forces in a microchannel by hybrid boundary element method and immersed boundary‐lattice Boltzmann method

Efficient pumping of blood flow in a microfluidic device is essential for rapid detection of bacterial bloodstream infections (BSI) using alternating current (AC) electrokinetics. Compared with AC electro‐osmosis (ACEO) phenomenon, the advantage of AC electrothermal (ACET) mechanism is its capability of pumping biofluids with high electrical conductivities at a relatively high AC voltage frequency. In the current work, the microfluidic pumping of non‐Newtonian blood flow using ACET forces is investigated in detail by modeling its multi‐physics process with hybrid boundary element method (BEM) and immersed boundary‐lattice Boltzmann method (IB‐LBM). The Carreau–Yasuda model is used to simulate the realistic rheological behavior of blood flow. The ACET pumping efficiency of blood flow is studied in terms of different AC voltage magnitudes and frequencies, thermal boundary conditions of electrodes, electrode configurations, channel height, and the channel length per electrode pair. Besides, the effect of rheological behavior on the blood flow velocity is theoretically analyzed by comparing with the Newtonian fluid flow using scaling law analysis under the same physical conditions. The results indicate that the rheological behavior of blood flow and its frequency‐dependent dielectric property make the pumping phenomenon of blood flow different from that of the common Newtonian aqueous solutions. It is also demonstrated that using a thermally insulated electrode could enhance the pumping efficiency dramatically. Besides, the results conclude that increasing the AC voltage magnitude is a more economical pumping approach than adding the number of electrodes with the same energy consumption when the Joule heating effect is acceptable.     

Numerical analysis of a dielectrophoresis field‐flow fractionation device for the separation of multiple cell types

In this study, a dielectrophoresis field‐flow fractionation device was analyzed using a numerical simulation method and the behaviors of a set of different cells were investigated. By reducing the alternating current frequency of the electrodes from the value used in the original setup configuration and increasing the number of exit channels, total discrimination in cell trajectories and subsequent separation of four cell types were achieved. Cells were differentiated based on their size and dielectric response that are represented in their real part of Clausius–Mossotti factor at different frequencies. A number of novel designs were also proposed based on the original setup configuration. It was seen that by reducing the length of the main channel and the number of electrodes at low frequencies and not changing the inlet flow velocities, cell separation was still achieved successfully, although with a slightly larger electrode voltage. The shorter main channel decreased the residence time for the cells on the chip and also reduced the overall size of the device—these were improvements over the original design. The obtained results can be used to analyze other cell types by knowing their size and dielectric properties to design geometries that can ensure separation.     

A dielectrophoretic continuous flow sorter using integrated microelectrodes coupled to a channel constriction

In this article, we propose a novel dielectrophoretic continuous flow sorter using planar micro electrodes coupled to a channel constriction. This design enables a high particle sorting efficiency at low voltages while relying on a simple fabrication and integration process. We have numerically simulated the AC electrokinetic effects and the fluid behavior to predict particle trajectories. Simulation results are in accordance with experimental data: 10 and 5 μm polystyrene beads were continuously sorted with <2% errors at flow speeds of 100 μm/s. We were also able to change the particle buffer while sorting beads. Finally, to demonstrate the interest of our device for cell sorting, we also sorted dead and living yeast cells according to their different dielectric properties. Living cell concentration was enriched by a factor of 4 versus dead cell concentration after passing the sorting device.     

Analytical solutions and validation of electric field and dielectrophoretic force in a bio-microfluidic channel

In a microbiological device, cell or particle manipulation and characterization require the use of electric field on different electrodes in several configurations and shapes. To efficiently design microelectrodes within a microfluidic channel for dielectrophoresis focusing, manipulation and characterization of cells, the designer will seek the exact distribution of the electric potential, electric field and hence dielectrophoresis force exerted on the cell within the microdevice. In this paper we describe the approach attaining the analytical solution of the dielectrophoretic force expression within a microchannel with parallel facing same size electrodes present on the two faces of channel substrates, with opposite voltages on the pair electrodes. Simple Fourier series mathematical expressions are derived for electric potential, electric field and dielectric force between two distant finite‐size electrodes. Excellent agreement is found by comparing the analytical results calculated using MATLAB? with numerical ones obtained by Comsol. This analytical result can help the designer to perform simple design parametric analysis. Bio‐microdevices are also designed and fabricated to illustrate the theoretical solution results with the experimental data. Experiments with red blood cells show the dielectrophoretic force contour plots of the analytical data matched to the experimental results.     

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.     

Size-resolved counting of circulating tumor cells on pinched flow-based microfluidic cytometry

Precise cell detecting and counting is meaningful in circulating tumor cells (CTCs) analysis. In this work, a simple cyclic olefin copolymer (COC) microflow cytometer device was developed for size-resolved CTCs counting. The proposed device is constructed by a counting channel and a pinched injection unit having three channels. Through injection flow rate control, microspheres/cells can be focused into the centerline of the counting channel. Polystyrene microspheres of 3, 9, 15, and 20 µm were used for the microspheres focusing characterization. After coupling to laser-induced fluorescence detection technique, the proposed device was used for polystyrene microspheres counting and sizing. A count accuracy up to 97.6% was obtained for microspheres. Moreover, the proposed microflow cytometer was applied to CTCs detecting and counting. To mimic blood sample containing CTCs and CTCs mixture with different subtypes, an MDA-MB-231 (human breast cell line) spiked red blood cells sample and a mixture of MDA-MB-231 and MCF-7 (human breast cell line) sample were prepared, respectively, and then analyzed by the developed pinched flow-based microfluidic cytometry. The simple fabricated and easy operating COC microflow cytometer exhibits the potential in the point-of-care clinical application.     

Parallel single-cell optical transit dielectrophoresis cytometer

In this work, we present an optical transit DEP flow cytometer for parallel single-cell analysis. Each cell's dielectric property is inferred from velocity perturbations due to DEP actuation in a microfluidic channel. Dual LED sources facilitate velocity measurement by producing two transit shadows for each cell passing through the channel. These shadows are detected using a 256-pixel linear optical array detector. Massively parallel analysis is possible as each pixel of the detector can independently analyze the passing cells. A wide channel (∼18 mm) was employed to carry many particles simultaneously, and the system was capable of detecting the velocity of over 200 cells simultaneously. We have achieved analysis rates for 10 µm diameter polystyrene spheres response exceeding 250 per second. With appropriate calibration, this DEP cytometer can quantitatively measure the dielectric response. The dielectric response (Clausius–Mossotti factor) of viable CHO cells was measured over the frequency range of 100 kHz to 6 MHz, and the obtained response matches the previously measured values by our group. The DEP cytometer uses simple modular components to achieve high throughput label-free single-cell dielectric analysis and can begin analyzing particles within 10 s after starting to pump the sample into the channel.     

Determination of magnetic susceptibility of various ion-labeled red blood cells by means of analytical magnetapheresis

Analytical magnetapheresis is a newly developed technique for separating magnetically susceptible particles. The magnetically susceptible particles are deposited on a bottom plate after flowing through a thin (< 0.05 cm) separation channel under a magnetic field applied perpendicular to the flow. Particles with various magnetic susceptibilities can be selectively deposited and separated by adjusting the applying magnetic force and flow rates. Magnetic susceptibility is an important parameter for magnetic separation. Magnetic susceptibility determination of various ion-labeled red blood cells (RBCs) using analytical magnetapheresis with a simple theoretical treatment is reported in this study. Susceptibility determination is based on the balance between maximal channel flow rate and magnetically induced flow rate for deposition. We tried a new approach to determine particle magnetic susceptibilities using a balance of magnetic and drag forces to control magnetically induced particle velocities. The Er3+, Fe3+, Cu2+, Mn2+, Co2+, and Ni2+ ions were used to label RBC at various labeling concentrations for susceptibility determination. The susceptibilities determined for various ion-labeled RBC under two magnetic field intensities fell within a 10% range. The average viabilities of various ion-labeled RBCs were 96.1 +/- 0.8%. The susceptibility determination generally took less than 10 min. Determined susceptibilities from analytical magnetapheresis differed by 10% from reference measurements using a superconducting quantum interference device (SQUID) magnetometer. The cost and time for analysis is much less using analytical magnetapheresis. This technique can provide a simple, fast, and economical way for particle susceptibility determinations.     

Dielectrophoretic sorting on a microfabricated flow cytometer: label free separation of Babesia bovis infected erythrocytes

Dielectrophoresis is a method that has demonstrated great potential in cell discrimination and isolation. In this study, the dielectrophoretic sorting of normal and Babesia bovis infected erythrocytes was performed using a microfabricated flow cytometer. Separation was possible through exploitation of the dielectric differences between normal and infected erythrocytes, essentially due to the higher ionic membrane permeability of B. bovis infected cells. Sorting experiments were performed inside a microchip made from Pt microelectrodes and SU-8 channels patterned on a glass substrate. Optimum cell separation was achieved at 4 MHz using an in vitro culture of B. bovis suspended in 63 mS/m phosphate buffer and applying a sinusoidal voltage of 15 V peak-to-peak. Normal erythrocytes experienced stronger positive dielectrophoresis (pDEP) than B. bovis infected cells, moving them closer to the microelectrodes. Under these conditions it was possible to enrich the fraction of infected cells from 7 to 50% without the need of extensive sample preparation or labelling. Throughout the experiments very few microliters of sample were used, suggesting that this system may be considered suitable for integration in a low-cost automated device to be used in the in situ diagnostic of babesiosis.     

Manipulation and characterization of red blood cells with alternating current fields in microdevices

The motion of a suspension of erythrocytes (red blood cells, RBCs) in response to a high-frequency alternating current (AC) field in a microfluidic device is examined with parallel and orthogonal electrode configurations to delineate the various fundamental driving forces. Cell repulsion from the platinum electrodes due to electrode polarization interacting with cell membrane polarizations is observed to be the strongest force acting on the particles in the first few seconds of field application. We exploit this strong repulsion to concentrate the bioparticles between the microelectrodes to amplify multiparticle aggregation phenomenon and dielectrophoretic (DEP) manipulation in a small and well-characterized region within the microfluidic device. Secondary motions include RBC pearl chain formation along field lines due to particle polarization followed by classical dielectrophoretic motion of the chains across field lines to regions of weaker field. These are driven by far weaker dipole-dipole and field-dipole interactions than the preliminary electrode repulsions. RBC chain length and total aggregated cells are presented for a variety of AC frequencies and are significantly amplified by the electrode repulsion. Motion of particles away from the polarized electrode is found to be species- and age-sensitive and can stand by itself as a promising identification and separation mechanism. In a 0.1 S/m isotonic phosphate buffer saline medium, we observe the largest cell mobilities at an optimal frequency of approximately 1 MHz, corresponding to the inverse diffusion time across the double layer of the cell and across the electrode's polarized layer. This suggests that the dielectric responses of both particles and electrodes in the low MHz frequency range are mostly determined by normal electromigration of ions from the bulk to their interfaces. Sensitivity to RBC age and species suggests that the surface proteins and membrane ion channels can affect the capacitance of the interface to accommodate the ions from the bulk. Such surface ion accumulation and polarization mechanisms are different from the classical dielectric theories. The resonant frequency of electrode polarization at around 1 MHz falls between positive and negative dielectrophoretic resonant frequency peaks - suggesting that the double-layer polarization mechanism is a distinct and potentially important bioparticle manipulation tool.     

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

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

Chromatographic behaviour of single cells in a microchannel with dynamic geometry

We present the design of a microchannel with dynamic geometry that imparts different flow rates to different cells based on their physical properties. This dynamic microchannel is formed between a textured surface and a flexible membrane. As cells flow through the microchannel, the height of the channel oscillates causing periodic entrapment of the larger cells, and as a result, attenuating their velocity relative to the bulk liquid. The smaller cells are not slowed by the moving microstructure, and move synchronously with the bulk liquid. The ability of the dynamic microchannel to selectively attenuate the flow rate of eukaryotic cells is similar to a size-exclusion chromatography column, but with the opposite behavior. The speed of smaller substances is attenuated relative to the larger substances in traditional size-exclusion chromatography columns, whereas the speed of the larger substances that is attenuated in the dynamic microchannel. We verified this property by tracking the flow of single cells through the dynamic microchannel. L1210 mouse lymphoma cells (MLCs), peripheral blood mononuclear cells (PBMCs), and red blood cells (RBCs) were used as model cells. We showed that the flow rate of MLC is slowed by more than 50% compared to PBMCs and RBCs. We characterized the operation of the microchannel by measuring the velocity of each of the three cell types as a function of the pressures used to oscillate the membrane position, as well as the duty cycle of the oscillation.     

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.     

A combined micromagnetic-microfluidic device for rapid capture and culture of rare circulating tumor cells

Here we describe a combined microfluidic-micromagnetic cell separation device that has been developed to isolate, detect and culture circulating tumor cells (CTCs) from whole blood, and demonstrate its utility using blood from mammary cancer-bearing mice. The device was fabricated from polydimethylsiloxane and contains a microfluidic architecture with a main channel and redundant 'double collection' channel lined by two rows of dead-end side chambers for tumor cell collection. The microdevice design was optimized using computational simulation to determine dimensions, magnetic forces and flow rates for cell isolation using epithelial cell adhesion molecule (EpCAM) antibody-coated magnetic microbeads (2.8 μm diameter). Using this device, isolation efficiencies increased in a linear manner and reached efficiencies close to 90% when only 2 to 80 breast cancer cells were spiked into a small volume (1.0 mL) of blood taken from wild type mice. The high sensitivity visualization capabilities of the device also allowed detection of a single cell within one of its dead-end side chambers. When blood was removed from FVB C3(1)-SV40 T-antigen mammary tumor-bearing transgenic mice at different stages of tumor progression, cells isolated in the device using anti-EpCAM-beads and magnetically collected within the dead-end side chambers, also stained positive for pan-cytokeratin-FITC and DAPI, negative for CD45-PerCP, and expressed SV40 large T antigen, thus confirming their identity as CTCs. Using this isolation approach, we detected a time-dependent rise in the number of CTCs in blood of female transgenic mice, with a dramatic increase in the numbers of metastatic tumor cells appearing in the blood after 20 weeks when tumors transition to invasive carcinoma and exhibit increased growth of metastases in this model. Importantly, in contrast to previously described CTC isolation methods, breast tumor cells collected from a small volume of blood removed from a breast tumor-bearing animal remain viable and they can be easily removed from these devices and expanded in culture for additional analytical studies or potential drug sensitivity testing.     

Characterization of particle capture in a sawtooth patterned insulating electrokinetic microfluidic device

Here we present a scheme to separate particles according to their characteristic physical properties, including size, charge, polarizability, deformability, surface charge mobility, dielectric features, and local capacitance. Separation is accomplished using a microdevice based on direct current insulator gradient dielectrophoresis that can isolate and concentrate multiple analytes simultaneously at different positions. The device is dependent upon dielectrophoretic and electrokinetic forces incorporating a global longitudinal direct current field as well as using shaped insulating features within the channel to induce local gradients. This design allows for the production of strong local field gradients along a global field causing particles to enter, initially transported through the channel by electrophoresis and electroosmosis (electrokinetics), and to be isolated via repulsive dielectrophoretic forces that are proportional to an exponent of the field gradient. Sulfate-capped polystyrene nano and microparticles (20, 200 nm, and 1 μm) were used as probes to demonstrate the influence of channel geometry and applied longitudinal field on separation behavior. These results are consistent with models using similar channel geometry and indicate that specific particulate species can be isolated within a distinct portion of the device, whereas concentrating particles by factors from 10(3) to 10(6).     

Double spiral microchannel for label-free tumor cell separation and enrichment

This work reports on a passive double spiral microfluidic device allowing rapid and label-free tumor cell separation and enrichment from diluted peripheral whole blood, by exploiting the size-dependent hydrodynamic forces. A numerical model is developed to simulate the Dean flow inside the curved geometry and to track the particle/cell trajectories, which is validated against the experimental observations and serves as a theoretical foundation for optimizing the operating conditions. Results from separating tumor cells (MCF-7 and Hela) spiked into whole blood indicate that 92.28% of blood cells and 96.77% of tumor cells are collected at the inner and the middle outlet, respectively, with 88.5% tumor recovery rate at a throughput of 3.33 × 10(7) cells min(-1). We expect that this label-free microfluidic platform, driven by purely hydrodynamic forces, would have an impact on fundamental and clinical studies of circulating tumor cells.     

Microfluidic system for dielectrophoretic separation based on a trapezoidal electrode array

This paper presents a novel microfluidic device for dielectrophoretic separation based on a trapezoidal electrode array (TEA). In this method, particles with different dielectric properties are separated by the device composed of the TEA for the dielectrophoretic deflection of particles under negative dielectrophoresis (DEP) and poly(dimethylsiloxane)(PDMS) microfluidic channel with a sinuous and expanded region. Polystyrene microparticles are exposed to an electric field generated from the TEA in the microfluidic channel and are dielectrophoretically focused to make all of them line up to one sidewall. When these particles arrive at the region of another TEA for dielectrophoretic separation, they are separated having different positions along the perpendicular direction to the fluid flow due to their different dielectrophoretic velocities. To evaluate the separation process and performance, both the effect of the flow rate on dielectrophoretic focusing and the influence of the number of trapezoidal electrodes on dielectrophoretic separation are investigated. Now that this method utilizes the TEA as a source of negative DEP, non-specific particle adhering to the electrode surface can be prevented; conventional separation approaches depending on the positive DEP force suffer from this problem. In addition, since various particle types are continuously separated, this method can be easily applicable to the separation and analysis of various dielectric particles with high particle recovery and selectivity.