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.     

Negative dielectrophoretic capture of bacterial spores in food matrices

A microfluidic device with planar square electrodes is developed for capturing particles from high conductivity media using negative dielectrophoresis (n-DEP). Specifically, Bacillus subtilis and Clostridium sporogenes spores, and polystyrene particles are tested in NaCl solution (0.05 and 0.225 S∕m), apple juice (0.225 S∕m), and milk (0.525 S∕m). Depending on the conductivity of the medium, the Joule heating produces electrothermal flow (ETF), which continuously circulates and transports the particles to the DEP capture sites. Combination of the ETF and n-DEP results in different particle capture efficiencies as a function of the conductivity. Utilizing 20 μm height DEP chambers, “almost complete” and rapid particle capture from lower conductivity (0.05 S∕m) medium is observed. Using DEP chambers above 150 μm in height, the onset of a global fluid motion for high conductivity media is observed. This motion enhances particle capture on the electrodes at the center of the DEP chamber. The n-DEP electrodes are designed to have well defined electric field minima, enabling sample concentration at 1000 distinct locations within the chip. The electrode design also facilitates integration of immunoassay and other surface sensors onto the particle capture sites for rapid detection of target micro-organisms in the future.     

Frequency sweep rate dependence on the dielectrophoretic response of polystyrene beads and red blood cells

Alternating current (AC) dielectrophoresis (DEP) experiments for biological particles in microdevices are typically done at a fixed frequency. Reconstructing the DEP response curve from static frequency experiments is laborious, but essential to ascertain differences in dielectric properties of biological particles. Our lab explored the concept of sweeping the frequency as a function of time to rapidly determine the DEP response curve from fewer experiments. For the purpose of determining an ideal sweep rate, homogeneous 6.08 μm polystyrene (PS) beads were used as a model system. Translatability of the sweep rate approach to ∼7 μm red blood cells (RBC) was then verified. An Au/Ti quadrapole electrode microfluidic device was used to separately subject particles and cells to 10Vpp AC electric fields at frequencies ranging from 0.010 to 2.0 MHz over sweep rates from 0.00080 to 0.17 MHz/s. PS beads exhibited negative DEP assembly over the frequencies explored due to Maxwell-Wagner interfacial polarizations. Results demonstrate that frequency sweep rates must be slower than particle polarization timescales to achieve reliable incremental polarizations; sweep rates near 0.00080 MHz/s yielded DEP behaviors very consistent with static frequency DEP responses for both PS beads and RBCs.     

Dielectrophoretic spectra of translational velocity and critical frequency for a spheroid in traveling electric field

An analysis has been made of the dielectrophoretic (DEP) forces acting on a spheroidal particle in a traveling alternating electric field. The traveling field can be generated by application of alternating current signals to an octapair electrode array arranged in phase quadrature sequence. The frequency dependent force can be resolved into two orthogonal forces that are determined by the real and the imaginary parts of the Clausius–Mossotti factor. The former is determined by the gradient in the electric field and directs the particle either toward or away from the tip of the electrodes in the electrode array. The force determined by the imaginary component is in a direction along the track of the octapair interdigitated electrode array. The DEP forces are related to the dielectric properties of the particle. Experiments were conducted to determine the DEP forces in such an electrode arrangement using yeast cells (Saccharomyces cervisiate TISTR 5088) with media of various conductivities. Experimental data are presented for both viable and nonviable cells. The dielectric properties so obtained were similar to those previously reported in literature using other DEP techniques.     

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).     

Optoelectrofluidic field separation based on light-intensity gradients

Optoelectrofluidic field separation (OEFS) of particles under light -intensity gradient (LIG) is reported, where the LIG illumination on the photoconductive layer converts the short-ranged dielectrophoresis (DEP) force to the long-ranged one. The long-ranged DEP force can compete with the hydrodynamic force by alternating current electro-osmosis (ACEO) over the entire illumination area for realizing effective field separation of particles. In the OEFS system, the codirectional illumination and observation induce the levitation effect, compensating the attenuation of the DEP force under LIG illumination by slightly floating particles from the surface. Results of the field separation and concentration of diverse particle pairs (0.82–16 μm) are well demonstrated, and conditions determining the critical radius and effective particle manipulation are discussed. The OEFS with codirectional LIG strategy could be a promising particle manipulation method in many applications where a rapid manipulation of biological cells and particles over the entire working area are of interest.     

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.     

Field-free particle focusing in microfluidic plugs

Particle concentration is a key unit operation in biochemical assays. Although there are many techniques for particle concentration in continuous-phase microfluidics, relatively few are available in multiphase (plug-based) microfluidics. Existing approaches generally require external electric or magnetic fields together with charged or magnetized particles. This paper reports a passive technique for particle concentration in water-in-oil plugs which relies on the interaction between particle sedimentation and the recirculating vortices inherent to plug flow in a cylindrical capillary. This interaction can be quantified using the Shields parameter (θ), a dimensionless ratio of a particle’s drag force to its gravitational force, which scales with plug velocity. Three regimes of particle behavior are identified. When θ is less than the movement threshold (region I), particles sediment to the bottom of the plug where the internal vortices subsequently concentrate the particles at the rear of the plug. We demonstrate highly efficient concentration (∼100%) of 38 μm glass beads in 500 μm diameter plugs traveling at velocities up to 5 mm/s. As θ is increased beyond the movement threshold (region II), particles are suspended in well-defined circulation zones which begin at the rear of the plug. The length of the zone scales linearly with plug velocity, and at sufficiently large θ, it spans the length of the plug (region III). A second effect, attributed to the co-rotating vortices at the rear cap, causes particle aggregation in the cap, regardless of flow velocity. Region I is useful for concentrating/collecting particles, while the latter two are useful for mixing the beads with the solution. Therefore, the two key steps of a bead-based assay, concentration and resuspension, can be achieved simply by changing the plug velocity. By exploiting an interaction of sedimentation and recirculation unique to multiphase flow, this simple technique achieves particle concentration without on-chip components, and could therefore be applied to a range of heterogeneous screening assays in discrete nl plugs.     

Characterizing the dielectric properties of human mesenchymal stem cells and the effects of charged elastin-like polypeptide copolymer treatment

Human mesenchymal stem cells (hMSCs) have three key properties that make them desirable for stem cell therapeutics: differentiation capacity, trophic activity, and ability to self-renew. However, current separation techniques are inefficient, time consuming, expensive, and, in some cases, alter hMSCs cellular function and viability. Dielectrophoresis (DEP) is a technique that uses alternating current electric fields to spatially separate biological cells based on the dielectric properties of their membrane and cytoplasm. This work implements the first steps toward the development of a continuous cell sorting microfluidic device by characterizing native hMSCs dielectric signatures and comparing them to hMSCs morphologically standardized with a polymer. A quadrapole Ti-Au electrode microdevice was used to observe hMSC DEP behaviors, and quantify frequency spectra and cross-over frequency of hMSCs from 0.010–35 MHz in dextrose buffer solutions (0.030 S/m and 0.10 S/m). This combined approach included a systematic parametric study to fit a core-shell model to the DEP spectra over the entire tested frequency range, adding robustness to the analysis technique. The membrane capacitance and permittivity were found to be 2.2 pF and 2.0 in 0.030 S/m and 4.5 pF and 4.1 in 0.10 S/m, respectively. Elastin-like polypeptide (ELP-) polyethyleneimine (PEI) copolymer was used to control hMSCs morphology to spheroidal cells and aggregates. Results demonstrated that ELP-PEI treatment controlled hMSCs morphology, increased experiment reproducibility, and concurrently increased hMSCs membrane permittivity to shift the cross-over frequency above 35 MHz. Therefore, ELP-PEI treatment may serve as a tool for the eventual determination of biosurface marker-dependent DEP signatures and hMSCs purification.     

Efficient manipulation of microparticles in bubble streaming flows

Oscillating microbubbles of radius 20–100 μm driven by ultrasound initiate a steady streaming flow around the bubbles. In such flows, microparticles of even smaller sizes (radius 1–5 μm) exhibit size-dependent behaviors: particles of different sizes follow different characteristic trajectories despite density-matching. Adjusting the relative strengths of the streaming flow and a superimposed Poiseuille flow allows for a simple tuning of particle behavior, separating the trajectories of particles with a size resolution on the order of 1 μm. Selective trapping, accumulation, and release of particles can be achieved. We show here how to design bubble microfluidic devices that use these concepts to filter, enrich, and preconcentrate particles of selected sizes, either by concentrating them in discrete clusters (localized both stream- and spanwise) or by forcing them into narrow, continuous trajectory bundles of strong spanwise localization.     

An integrated dielectrophoretic chip for continuous bioparticle filtering, focusing, sorting, trapping, and detecting

Multi-target pathogen detection using heterogeneous medical samples require continuous filtering, sorting, and trapping of debris, bioparticles, and immunocolloids within a diagnostic chip. We present an integrated AC dielectrophoretic (DEP) microfluidic platform based on planar electrodes that form three-dimensional (3D) DEP gates. This platform can continuously perform these tasks with a throughput of 3 μL∕min. Mixtures of latex particles, Escherichia coli Nissle, Lactobacillus, and Candida albicans are sorted and concentrated by these 3D DEP gates. Surface enhanced Raman scattering is used as an on-chip detection method on the concentrated bacteria. A processing rate of 500 bacteria was estimated when 100 μl of a heterogeneous colony of 10⁷ colony forming units ∕ml was processed in a single pass within 30 min.     

Dielectrophoretic capture of low abundance cell population using thick electrodes

Enrichment of rare cell populations such as Circulating Tumor Cells (CTCs) is a critical step before performing analysis. This paper presents a polymeric microfluidic device with integrated thick Carbon-PolyDimethylSiloxane composite (C-PDMS) electrodes designed to carry out dielectrophoretic (DEP) trapping of low abundance biological cells. Such conductive composite material presents advantages over metallic structures. Indeed, as it combines properties of both the matrix and doping particles, C-PDMS allows the easy and fast integration of conductive microstructures using a soft-lithography approach while preserving O₂ plasma bonding properties of PDMS substrate and avoiding a cumbersome alignment procedure. Here, we first performed numerical simulations to demonstrate the advantage of such thick C-PDMS electrodes over a coplanar electrode configuration. It is well established that dielectrophoretic force (F_DEP) decreases quickly as the distance from the electrode surface increases resulting in coplanar configuration to a low trapping efficiency at high flow rate. Here, we showed quantitatively that by using electrodes as thick as a microchannel height, it is possible to extend the DEP force influence in the whole volume of the channel compared to coplanar electrode configuration and maintaining high trapping efficiency while increasing the throughput. This model was then used to numerically optimize a thick C-PDMS electrode configuration in terms of trapping efficiency. Then, optimized microfluidic configurations were fabricated and tested at various flow rates for the trapping of MDA-MB-231 breast cancer cell line. We reached trapping efficiencies of 97% at 20 μl/h and 78.7% at 80 μl/h, for 100 μm thick electrodes. Finally, we applied our device to the separation and localized trapping of CTCs (MDA-MB-231) from a red blood cells sample (concentration ratio of 1:10).     

Microfluidic sorting of microtissues

Increasingly, invitro culture of adherent cell types utilizes three-dimensional (3D) scaffolds or aggregate culture strategies to mimic tissue-like, microenvironmental conditions. In parallel, new flow cytometry-based technologies are emerging to accurately analyze the composition and function of these microtissues (i.e., large particles) in a non-invasive and high-throughput way. Lacking, however, is an accessible platform that can be used to effectively sort or purify large particles based on analysis parameters. Here we describe a microfluidic-based, electromechanical approach to sort large particles. Specifically, sheath-less asymmetric curving channels were employed to separate and hydrodynamically focus particles to be analyzed and subsequently sorted. This design was developed and characterized based on wall shear stress, tortuosity of the flow path, vorticity of the fluid in the channel, sorting efficiency and enrichment ratio. The large particle sorting device was capable of purifying fluorescently labelled embryoid bodies (EBs) from unlabelled EBs with an efficiency of 87.3% ± 13.5%, and enrichment ratio of 12.2 ± 8.4 (n = 8), while preserving cell viability, differentiation potential, and long-term function.     

Manipulating particle trajectories with phase-control in surface acoustic wave microfluidics

We present a 91 MHz surface acoustic wave resonator with integrated microfluidics that includes a flow focus, an expansion region, and a binning region in order to manipulate particle trajectories. We demonstrate the ability to change the position of the acoustic nodes by varying the electronic phase of one of the transducers relative to the other in a pseudo-static manner. The measurements were performed at room temperature with 3 μm diameter latex beads dispersed in a water-based solution. We demonstrate the dependence of nodal position on pseudo-static phase and show simultaneous control of 9 bead streams with spatial control of −0.058 μm/deg ± 0.001 μm/deg. As a consequence of changing the position of bead streams perpendicular to their flow direction, we also show that the integrated acoustic-microfluidic device can be used to change the trajectory of a bead stream towards a selected bin with an angular control of 0.008 deg/deg ± 0.000(2) deg/deg.     

Three dimensional passivated-electrode insulator-based dielectrophoresis

In this study, a 3D passivated-electrode, insulator-based dielectrophoresis microchip (3D πDEP) is presented. This technology combines the benefits of electrode-based DEP, insulator-based DEP, and three dimensional insulating features with the goal of improving trapping efficiency of biological species at low applied signals and fostering wide frequency range operation of the microfluidic device. The 3D πDEP chips were fabricated by making 3D structures in silicon using reactive ion etching. The reusable electrodes are deposited on second glass substrate and then aligned to the microfluidic channel to capacitively couple the electric signal through a 100 μm glass slide. The 3D insulating structures generate high electric field gradients, which ultimately increases the DEP force. To demonstrate the capabilities of 3D πDEP, Staphylococcus aureus was trapped from water samples under varied electrical environments. Trapping efficiencies of 100% were obtained at flow rates as high as 350 μl/h and 70% at flow rates as high as 750 μl/h. Additionally, for live bacteria samples, 100% trapping was demonstrated over a wide frequency range from 50 to 400 kHz with an amplitude applied signal of 200 Vpp. 20% trapping of bacteria was observed at applied voltages as low as 50 Vpp. We demonstrate selective trapping of live and dead bacteria at frequencies ranging from 30 to 60 kHz at 400 Vpp with over 90% of the live bacteria trapped while most of the dead bacteria escape.     

Dielectrophoretic manipulation of particles in a modified microfluidic H filter with multi-insulating blocks

The conventional microfluidic H filter is modified with multi-insulating blocks to achieve a flow-through manipulation and separation of microparticles. The device transports particles by exploiting electro-osmosis and electrophoresis, and manipulates particles by utilizing dielectrophoresis (DEP). Polydimethylsiloxane (PDMS) blocks fabricated in the main channel of the PDMS H filter induce a nonuniform electric field, which exerts a negative DEP force on the particles. The use of multi-insulating blocks not only enhances the DEP force generated, but it also increases the controllability of the motion of the particles, facilitating their manipulation and separation. Experiments were conducted to demonstrate the controlled flow direction of particles by adjusting the applied voltages and the separation of particles by size under two different input conditions, namely (i) a dc electric field mode and (ii) a combined ac and dc field mode. Numerical simulations elucidate the electrokinetic and hydrodynamic forces acting on a particle, with theoretically predicted particle trajectories in good agreement with those observed experimentally. In addition, the flow field was obtained experimentally with fluorescent tracer particles using the microparticle image velocimetry (μ-PIV) technique.     

Continuous size-based separation of microparticles in a microchannel with symmetric sharp corner structures

A new microchannel with a series of symmetric sharp corner structures is reported for passive size-dependent particle separation. Micro particles of different sizes can be completely separated based on the combination of the inertial lift force and the centrifugal force induced by the sharp corner structures in the microchannel. At appropriate flow rate and Reynolds number, the centrifugal force effect on large particles, induced by the sharp corner structures, is stronger than that on small particles; hence after passing a series of symmetric sharp corner structures, large particles are focused to the center of the microchannel, while small particles are focused at two particle streams near the two side walls of the microchannel. Particles of different sizes can then be completely separated. Particle separation with this device was demonstrated using 7.32 μm and 15.5 μm micro particles. Experiments show that in comparison with the prior multi-orifice flow fractionation microchannel and multistage-multiorifice flow fractionation microchannel, this device can completely separate two-size particles with narrower particle stream band and larger separation distance between particle streams. In addition, it requires no sheath flow and complex multi-stage separation structures, avoiding the dilution of analyte sample and complex operations. The device has potentials to be used for continuous, complete particle separation in a variety of lab-on-a-chip and biomedical applications.     

Influences of electric field on living cells in a charged water-in-oil droplet under electrophoretic actuation

We experimentally investigate the effects of high electric field on living cells inside a charged droplet under electrophoretic actuation. When an aqueous droplet suspended in a dielectric liquid contacts with electrified electrode, the droplet acquires charge. This charged droplet undergoes electrophoretic motion under strong electric field (1–3 kV/cm), which can be used as a droplet manipulation method in biomicrofluidic applications. However, because strong electric field and use of dielectric oil can be a harmful environment for living cells, the biological feasibilities have been tested. Trypan blue test and cell growth test have been performed to check the viability and proliferation of cells in a droplet under various electric field strengths and actuation times. We have not observed any noticeable influence of electric field and silicone oil on the viability and proliferation of cells, which indicates that electrophoresis could be safely used as a manipulation method for a droplet containing living biological system.     

Cell separation and transportation between two miscible fluid streams using ultrasound

This paper presents a two-stream microfluidic system for transporting cells or micro-sized particles from one fluid stream to another by acoustophoresis. The two fluid streams, one being the original suspension and the other being the destination fluid, flow parallel to each other in a microchannel. Using a half-wave acoustic standing wave across the channel width, cells or particles with positive acoustic contrast factors are moved to the destination fluid where the pressure nodal line lies. By controlling the relative flow rate of the two fluid streams, the pressure nodal line can be maintained at a specific offset from the fluid interface within the destination fluid. Using this transportation method, particles or cells of different sizes and mechanical properties can be separated. The cells experiencing a larger acoustic radiation force are separated and transported from the original suspension to the destination fluid stream. The other particles or cells experiencing a smaller acoustic radiation force continue flowing in the original solution. Experiments were conducted to demonstrate the effective separation of polystyrene microbeads of different sizes (3 μm and 10 μm) and waterborne parasites (Giardia lamblia and Cryptosporidium parvum). Diffusion occurs between the two miscible fluids, but it was found to have little effects on the transport and separation process, even when the two fluids have different density and speed of sound.     

Microfluidic dielectrophoretic sorter using gel vertical electrodes

We report the development and results of a two-step method for sorting cells and small particles in a microfluidic device. This approach uses a single microfluidic channel that has (1) a microfabricated sieve which efficiently focuses particles into a thin stream, followed by (2) a dielectrophoresis (DEP) section consisting of electrodes along the channel walls for efficient continuous sorting based on dielectric properties of the particles. For our demonstration, the device was constructed of polydimethylsiloxane, bonded to a glass surface, and conductive agarose gel electrodes. Gold traces were used to make electrical connections to the conductive gel. The device had several novel features that aided performance of the sorting. These included a sieving structure that performed continuous displacement of particles into a single stream within the microfluidic channel (improving the performance of downstream DEP, and avoiding the need for additional focusing flow inlets), and DEP electrodes that were the full height of the microfluidic walls (“vertical electrodes”), allowing for improved formation and control of electric field gradients in the microfluidic device. The device was used to sort polymer particles and HeLa cells, demonstrating that this unique combination provides improved capability for continuous DEP sorting of particles in a microfluidic device.