Dielectrophoresis Crossover Frequency Research Articles

Dielectrophoretic profiling of erythrocytes to study the impacts of metabolic stress, temperature, and storage duration utilizing a point-and-planar microdevice

Dielectrophoresis (DEP) is widely utilized for trapping and sorting various types of cells, including live and dead cells and healthy and infected cells. This article focuses on the dielectric characterization of erythrocytes (red blood cells or RBCs) by quantifying DEP crossover frequency using a novel point-and-planar microwell device platform. Numerical simulations using COMSOL Multiphysics software demonstrate that the distribution of the DEP force is influenced by factors such as the shape of the point electrode, spacing between the point and planar electrodes, and the type of bioparticle being investigated. The dependency on electrode spacing is experimentally evaluated by analyzing the DEP crossover response of erythrocytes. Furthermore, the results are validated against the traditional electrical characterization technique called electrorotation, which typically requires laborious fabrication and operation using quadrupole electrodes. Other significant factors, including erythrocyte storage age and the changes in cell properties over time since collection, osmolarity, and temperature, are also assessed to determine the optimal conditions for erythrocyte characterization. The findings indicate a significant difference between fresh and stored erythrocyte samples (up to 4 days), highlighting the importance of maintaining an isotonic medium for cell storage.

Study on the dielectrophoretic characteristics of malaria-infected red blood cells.

Malaria is a tropical disease caused by parasites in the genus Plasmodium, which still presents 241 million cases and nearly 627,000 deaths recently. In this work, we used the dielectrophoresis (DEP) to characterize red blood cells in a microchannel. The purpose of this work is to determine the difference between the normal and the malaria-infected cells based on the DEP characteristics. The samples were infected cells and normal red blood cells, which were either prepared in culture or obtained from volunteers. Diamond-shaped and curved micropillars were used to create different degrees of DEP in the gap between them. The DEP crossover frequencies were observed with the diamond-shaped micropillars. The cell velocity under negative dielectrophoresis (nDEP) at a low frequency was examined with the curved micropillars. The measured lower crossover frequencies were remarkably different between the malaria-infected cells and the normal cells, whereas the higher crossover frequencies were similar among the samples. The velocity under nDEP was lower for the infected cells than the normal cells. The results imply that the malaria infection significantly decreases the capacitance but increases the conductance of the cell membrane, whereas a change in cytoplasmic conductivity may occur in a later stage of infection.

Self-aligned sequential lateral field non-uniformities over channel depth for high throughput dielectrophoretic cell deflection.

Dielectrophoresis (DEP) enables the separation of cells based on subtle subcellular phenotypic differences by controlling the frequency of the applied field. However, current electrode-based geometries extend over a limited depth of the sample channel, thereby reducing the throughput of the manipulated sample (sub-μL min-1 flow rates and <105 cells per mL). We present a flow through device with self-aligned sequential field non-uniformities extending laterally across the sample channel width (100 μm) that are created by metal patterned over the entire depth (50 μm) of the sample channel sidewall using a single lithography step. This enables single-cell streamlines to undergo progressive DEP deflection with minimal dependence on the cell starting position, its orientation versus the field and intercellular interactions. Phenotype-specific cell separation is validated (>μL min-1 flow and >106 cells per mL) using heterogeneous samples of healthy and glutaraldehyde-fixed red blood cells, with single-cell impedance cytometry showing that the DEP collected fractions are intact and exhibit electrical opacity differences consistent with their capacitance-based DEP crossover frequency. This geometry can address the vision of an "all electric" selective cell isolation and cytometry system for quantifying phenotypic heterogeneity of cellular systems.

Full Beta-Dispersion Region Dielectric Spectra and Dielectric Models of Viable and Non-Viable CHO Cells

The dielectric properties of biological cells can be used to gain information on their physiology and morphology. This paper reports the first measurements of the dielectric spectra of viable and non-viable cells over the full beta-dispersion (interfacial) frequency range. Dielectrophoresis (DEP) single cell in-flow techniques were employed to quantitatively measure the Clausius-Mossotti factor spectrum of individual cells over the 300 kHz–400 MHz range, covering both the MF and UHF DEP cross-over frequencies. Experiments were performed on Chinese hamster ovary (CHO) cells, one set cultured in growth media, the other in nutrient depleted media to induce apoptotic cells. Both cell states were measured using multi-frequency DEP flow cytometry, which provides the equivalent complex dielectric permittivity of individual cells. The measured dielectric spectra facilitate determination of a cell's morphology and the dielectric properties of its intracellular compartments, and are used to develop multi-shell dielectric models of viable and non-viable cells. The developed dielectric models can aid in biosensor design, in interpretation of bulk biomedia measurements where the heterogeneity in cell population can be masked, and in relating measured dielectric responses of cells to stimuli with changes in cellular physiology.

Application of dielectrophoresis towards characterization of rare earth elements biosorption by Cupriavidus necator

This work presents the dielectric characterization of rare earth elements (REEs) biosorption by Cupriavidus necator using dielectrophoretic crossover frequency measurements. Traditional means of characterizing biomass for biosorption is limited and time consuming. In this research we are presenting, for the first time, an electrokinetic method termed as dielectrophoresis (DEP) for the characterization of biosorption (uptake) of rare earth elements (REEs) by gram negative bacteria - Cupriavidus necator. To characterize, a 3mm-diameter point and planar microwell device platform is used to measure the DEP crossover frequency that yields the dielectric properties of the targeted biosorbents. Quantified dielectric properties of native Cupriavidus necator (REE−) and those exposed to rare earth elements (REE+): europium, neodymium, and samarium revealed a substantial change in the surface characteristics of the Cupriavidus necator after exposure to the REE solution. The response of C. necator to changes in REE exposure is substantially different for europium but similar between neodymium and samarium. Statistically both the REE+ and REE− groups dielectric signatures were significantly different proving that the REEs were absorbed by the bacteria. This research will revolutionize and impact the researchers and industrialists in the field of biosorption seeking for economical, greener, and sustainable means to recover REEs.

UHF-Dielectrophoresis Crossover Frequency as a New Marker for Discrimination of Glioblastoma Undifferentiated Cells

This paper introduces the first results of dielectric spectroscopy characterization of glioblastoma cells, measuring their crossover frequencies in the ultra-high-frequency range (above 50 MHz) by dielectrophoresis (DEP) techniques. Experiments were performed on two glioblastoma lines U87-MG and LN18 that were cultured following different conditions, in order to achieve different phenotypic profiles. We demonstrate here that the presented DEP electrokinetic method can be used to discriminate the undifferentiated from the differentiated cells. In this study, microfluidic lab-on-chip systems implemented on bipolar-complementary oxide semiconductor technology are used allowing single cell handling and analysis. Based on the characterizations of their own intracellular features, both the selected glioblastoma (GBM) cell lines cultured in distinct culture conditions have shown clear differences of DEP crossover frequency signatures compared to the differentiated cells cultured in a normal medium. These results support the concept and validate the efficiency for cell characterization in glioblastoma pathology.

Low-frequency dielectrophoretic response of a single particle in aqueous suspensions

We use optical tweezers-based dielectrophoresis (DEP) force spectroscopy to investigate the roles of the electrical double layer in the AC dielectric response of an individual colloidal particle in an aqueous medium. Specifically, we measure the DEP crossover frequency as a function of particles size, medium viscosity, and temperature. Experimental results were compared to low frequency relaxation mechanisms predicted by Schwarz, demonstrating the dielectrophoretic responses in the frequency range between 10 kHz and 1 MHz were dominated by counterion diffusion within the electric double layer.

Size-dependent dielectrophoretic crossover frequency of spherical particles.

Dielectrophoresis (DEP) has been extensively used in lab-on-a-chip systems for trapping, separating, and manipulating of micro particles suspended in a liquid medium. The most widely used analytic model, the dipole model, provides an accurate prediction on the crossover frequency of submicron particles, but cannot explain the significant drop in crossover frequency of larger particles. Here, we present numerical simulations using the Maxwell stress tensor (MST) and finite element method to study the size effect of the DEP crossover frequency of spherical polystyrene particles suspended in de-ionized water. Our results show that the surface conductance due to the electrical double layer plays a key role, and the size dependency of crossover frequency obtained by the MST method agrees reasonably well with published experimental data. The exponents of the power law are approximately -1.0 and -4.3 for smaller (diameter < 4.6 μm) and larger particles (diameter > 4.6 μm), respectively. The free surface charge distribution reveals that the charge begins accumulating on the particle equator for particle diameters larger than a critical diameter of 4.6 μm, a result not captured by the dipolar approximation. This method may be extended to analyze bioparticles with complex shapes and composition, and provides new insights into the interpretation of dielectrophoresis applications using lab-on-a-chip systems.

Discriminating dengue-infected hepatic cells (WRL-68) using dielectrophoresis.

Dielectrophoresis (DEP), the induced movement of dielectric particles placed in a nonuniform electric field, has been used as a potential technique for manipulation and separation of many biological samples without destructive consequences to the cell. Cells of the same genotype in different physiological and pathological states have unique morphological and structural features, therefore, it is possible to differentiate between them using their DEP responses. This paper reports the experimental discrimination of normal and dengue-infected human hepatic fetal epithelial cells (WRL-68 cells) based on their DEP crossover frequency, at which no resultant movement occurs in the cells in response to the DEP force. A microarray dot electrode was used to conduct the DEP experiments. The DEP forces applied to the cells were quantified by analyzing the light intensity shift within the electrode's dot region based on the Cumulative Modal Intensity Shift image analysis technique. The differences in dielectric properties between infected and uninfected cells were exploited by plotting a unique DEP spectrum for each set of cells. We observed that the crossover frequency decreased from 220 kHz for the normal WRL-68 cells to 140 kHz after infection with the dengue virus in a medium conductivity of 100 μS/cm. We conclude that the change in the DEP crossover frequency between dengue-infected cells and their healthy counterparts should allow direct characterization of these cell types by exploiting their electrophysiological properties.

Separation and assisted patterning of hippocampal neurons from glial cells using positive dielectrophoresis.

In this work, we describe the separation of embryonic mouse hippocampal neurons from glial cells using a positive dielectrophoresis (DEP) process. Here, we have implemented a cell trapping-favorable, cell suspension solution with low conductivity. It enables positive dielectrophoresis for hippocampal neurons (thereby attracting them to the electrodes), while resulting in negative dielectrophoresis for glial cells (repelling them from the electrodes). We have systematically performed a mathematical simulation and analysis to anticipate the DEP frequency at which hippocampal neurons and glial cells are separated. Simulated DEP crossover frequencies have been experimentally verified, and new, refined glial dielectric and physical properties are suggested that better reflect the experimental results obtained. DEP movements of neurons and glial cells in targeted separation media were experimentally analyzed, under the specified electric signal. Additionally, we have confirmed our modeling results by selectively trapping neurons over electrodes on a custom-made, multi-electrode array (MEA), resulting in active recruitment of neurons over the stimulation and recording sites. This technique is a valuable addition to the toolbox for creating more functional and versatile multi-electrode arrays.

(Keynote) Dielectrophoresis: Exploring the ‘2nd Frontier’ of Its Application in the Biomedical Sciences

Dielectrophoresis (DEP) has been widely studied for its potential as a biomarker-free method of sorting cells based on their intrinsic dielectric properties [1-4]. Under appropriate experimental conditions the DEP frequency spectrum typically exhibited by a viable mammalian cell in suspension is characterized by two frequencies, f xo1 and f xo2. At these two characteristic frequencies the effective conductance and capacitance values of the cell exactly match those of the fluid it has displaced, and are commonly known as the DEP cross-over frequencies. At low frequencies (<10 kHz) viable cells typically exhibit negative DEP and move away from electrodes, with the transition to positive DEP occurring at f xo1 - where the cells move towards high field regions at the electrodes. The investigation, understanding (in terms of cell biology) and biomedical exploitation of this phenomenon can be considered to represent exploration of the ‘1st frontier of dielectrophoresis’. The results of this work, extending over more than 25 years, are now exploited in the form of various commercial devices for clinical and biomedical applications. The status of this activity will be reviewed. A transition back to negative DEP occurs at f xo2 (~150 MHz). The factors that control the high frequency DEP cross-over at f xo2 have only recently been explored, and the status and implications of this ‘2nd frontier of dielectrophoresis’ will also be reviewed in this presentation. 1. Pethig, R. Review Article - Dielectrophoresis: Status of the theory, technology, and applications. Biomicrofluidics 4, 022811 (2010). 2. Gagnon, Z.R. Cellular dielectrophoresis: Applications to the characterization, manipulation, separation and patterning of cells. Electrophoresis 32: 2466-2487 (2011). 3. Pethig, R. Dielectrophoresis: An assessment of its potential to aid the research and practice of drug discovery and delivery. Adv. Drug Deliv. Rev. 65: 1589-1599 (2013). 4. Gascoyne, P.R.C. and Shim, S. Isolation of circulating tumor cells by dielectrophoresis, Cancers 6: 545-579 (2014). 5. Chung, C., et al. Dielectrophoretic characterisation of mammalian cells above 100 MHz, J. Elec. Bioimpedance 2: 64-71 (2011)

Advances in Dielectrophoresis As a Biotechnology Tool

Dielectrophoresis (DEP) has been widely studied for its potential as a biomarker-free method of sorting cells based on their intrinsic dielectric properties. Under appropriate experimental conditions the DEP frequency spectrum typically exhibited by a viable mammalian cell in suspension is characterised by two frequencies, f xo1 and f xo2. At these two characteristic frequencies the effective conductance and capacitance values of the cell exactly match those of the fluid it has displaced, and are commonly known as the DEP cross-over frequencies. At low frequencies (< 10 kHz) viable cells typically exhibit negative DEP and move away from electrodes, with the transition to positive DEP occurring at f xo1 - where the cells move towards high field regions at the electrodes. A transition back to negative DEP occurs at f xo2 (~150 MHz). The dielectric properties of the cell membrane and cell interior can be evaluated from knowledge of the dielectric properties of the suspending fluid, and changes of the physico-chemical properties of the suspending fluid can be used to control the frequencies f xo1 and f xo2. Theoretical and experimental evaluations of f xo1 have been extensive and exploited in practical applications of DEP [1-5]. The current status of this, both in research and commercial exploitation, will be reviewed in this presentation. The factors that control the high frequency DEP cross-over at f xo2 are now under investigation. The status of the ‘exploration of this 2nd frontier of DEP research’ will be described. 1. Pethig, R. Review Article - Dielectrophoresis: Status of the theory, technology, and applications. Biomicrofluidics 4, 022811 (2010).2. Pethig, R.; Menachery, A.; Pells, S.; de Sousa, P. Dielectrophoresis: A review of applications for stem cell research. J. Biomed. Biotechnol., e182581 (2010).3. Gagnon, Z.R. Cellular dielectrophoresis: Applications to the characterization, manipulation, separation and patterning of cells. Electrophoresis 32: 2466-2487 (2011).4. Pethig, R. Dielectrophoresis: An assessment of its potential to aid the research and practice of drug discovery and delivery. Adv. Drug Deliv. Rev. 65: 1589-1599 (2013).5. Gascoyne, P.R.C. and Shim, S. Isolation of circulating tumor cells by dielectro-phoresis, Cancers 6: 545-579 (2014).

AC-electrokinetic manipulation and controlled encapsulate release of surfactant based micelles

Effective manipulation and understanding of the dynamic responses of complex micelle nanocolloids under ac-electric fields is of great scientific and technological importance due to the emerging interest in using rich electrokinetic phenomena for medical diagnostics and drug delivery. In this work, we employ fluorescence correlation spectroscopy (FCS) to study the ac-electrokinetic behaviors of surfactant-based micelles encapsulated with varied fluorescence and model drug probes at a single micelle level. Under applied non-uniform ac-fields of constant voltage across two adjacent microelectrodes in a quadrupole configuration, probe-encapsulated micelles exhibit positive dielectrophoresis (DEP) characteristics at the ac-frequency range of 10–500 kHz, in contrast to negative or zero DEP at higher frequency of approximately greater than 500 kHz. The transition from positive to negative or zero DEP is indicated by the field-induced decrease of micelle concentration in the FCS focal volume located in the high field regions between two adjacent microelectrodes. The estimated DEP crossover frequency from ω-dependent micelle concentration profile agrees with advanced DEP theory, which predicts the scaling of crossover frequency with Stern layer conductance. At a lower frequency range of ω = 5–10 kHz, unique and surprising response in the measured fluorescence autocorrelation functions of charged probe-encapsulated micelles is observed, but not so with neutral encapsulated probes, suggesting ac-field induced micelle instability with the resulting release of interior charged probe encapsulates. Intriguingly, the release of encapsulates from micelles by non-uniform ac-electric fields shows strong dependence on ac-frequency and encapsulate chemistry.

Refinement of the theory for extracting cell dielectric properties from dielectrophoresis and electrorotation experiments.

A modified theory is proposed for extracting cell dielectric properties from the peak frequency measurement of electrorotation (ER) and the crossover frequency measurement of dielectrophoresis (DEP). Current theory in the literature is based on the low frequency (DC) approximations for the equivalent cell permittivity and conductivity, which are valid when the measurements are performed in a medium with conductivity less than 1 mS/m. The present theory extracts the cell properties through optimizing an expression for the medium conductivity in terms of the peak ER, or DEP crossover, frequency according to its definition using full expressions of equivalent cell permittivity and conductivity. Various levels of approximation of the theory are proposed and discussed through a scaling analysis. The present theory can extract both membrane and interior properties from the low and the high peak ER, or DEP crossover, frequencies for any medium conductivity provided the peak ER, or DEP crossover, frequency exists. It can be reduced to the linear theory for the low peak ER and DEP crossover frequencies in the literature when the medium conductivity is less than 10 mS/m. However, we can determine the membrane capacitance and conductance via the slope and intercept, respectively, of the straight line fitting of the ER peak and DEP frequency against medium conductivity data according to the linear theory only when the intercept dominates the experimental uncertainty, which occurs when the medium conductivity is less than 1 mS/m in practice.

Biophysical Characteristics Reveal Neural Stem Cell Differentiation Potential

BackgroundDistinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.Methodology/Principal FindingsWe used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates.Conclusions/SignificanceWe show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

Dielectrophoresis of Janus particles under high frequency ac-electric fields

Janus polystyrene particles with gold and dielectric multilayer coatings on one hemisphere are shown to drastically alter the dielectrophoresis (DEP) behavior of homogeneous precursor particles under ac-electric fields. Alkanethiol coatings on the gold-coated hemisphere can effectively modify the DEP crossover frequency (ωc) with a negative (n) to positive (p) (p→n) DEP transition as increasing ac-frequency, in contrast to p→n DEP transition observed with precursor particles. All measured n→p DEP crossover frequencies with dependence on particle size, alkanethiol thickness, and medium conductivity are collapsed to yield a scaling with the resistance-capacitance (RC) time of the alkanethiol layer capacitance and the conductive medium resistance.

Designing a sensitive and quantifiable nanocolloid assay with dielectrophoretic crossover frequencies

Dielectrophoretic nanocolloid assay is a promising technique for sensitive molecular detection and identification, as target molecule hybridization onto the probe-functionalized nanocolloids can change their surface conductance and consequently their dielectrophoretic crossover frequencies. Thus, instead of relying on surface charge density increase after hybridization, as in many capacitive and field effect transistor impedance sensing techniques, the current assay utilizes the much larger surface conductance (and dielectrophoresis crossover frequency) changes to effect sensitive detection. Herein, we present a Poisson-Boltzmann theory for surfaces with finite-size molecular probes that include the surface probe conformation, their contribution to surface charge with a proper delineation of the slip and Stern planes. The theory shows that the most sensitive nanocolloid molecular sensor corresponds to a minimum in the dielectrophoretic crossover frequency with respect to the bulk concentration of the molecular probes (oligonucleotides in our case) during nanocolloid functionalization. This minimum yields the lowest number of functionalized probes that are also fully stretched because of surface probe-probe interaction. Our theory provides the surface-bulk oligonucleotide concentration isotherm and a folding number for the surface oligonucleotide conformation from the crossover frequency, the zeta potential, and the hydrodynamic radius data.

Optimized DNA hybridization detection on nanocolloidal particles by dielectrophoresis

Oligonucleotides of varying surface coverage are functionalized onto the surface of 100 nm silica particles and the corresponding hybridization reaction with target ssDNA is studied using dielectrophoresis (DEP). The measured DEP cross-over frequency (cof) is found to be sensitive to the oligonucleotide surface conformation. Zeta potential and particle size measurements suggest that at low oligo surface concentrations, non-specific binding of oligo to the particle surface prevents efficient hybridization. At high surface coverage, steric hindrance due to the fully stretched, tightly packed oligo conformation prevents diffusion of DNA molecules to the particle surface. The optimum surface coverage exists at intermediate coverage where the particle is found to be the least electrically conductive, and hence exhibits the lowest measured cof. A simple DEP cof measurement hence allows one to determine the optimal oligo surface coverage for increased hybridization efficiency and detection sensitivity.

Dielectrophoretic detection of membrane morphology changes in Jurkat T-cells undergoing etoposide-induced apoptosis

A dielectrophoresis (DEP) cell profiler, with concurrent FACS measurements, was used to monitor the morphological changes of Jurkat T-cells as they progressed through chemically induced apoptosis using etoposide. The cell 'physiometry' profiling technique measures the radius and the so-called DEP crossover frequency f(xo) of individual cells in a suspension, and this information was used to determine the effective plasma membrane capacitance of each cell. Control cells (n=526) exhibited a dynamic spread of f(xo) values, ranging from 50 to 250 kHz, and as apoptosis progressed over 6 h, the upper value for f(xo) progressively increased and extended beyond 500 kHz. This corresponded to a reduction in plasma membrane capacitance from 13.34 (+/-2.88) to 10.49 (+/-4.00) mF/m(2), and reflected a general smoothing of the membrane through loss of microvilli, for example. This is in broad agreement with previously reported studies of HL-60 cells undergoing apoptosis, but the authors' observation of a dynamic spread of f(xo) values does not agree with the earlier report that the f(xo) values for viable and apoptotic cells fall into two separable, relatively narrow, frequency bands. This has implications when devising protocols for the efficient DEP separation of viable, apoptotic and necrotic cells.

Cell Physiometry Tools Based on Dielectrophoresis

A cell physiometry system is described for characterizing and separating cells, and performing cell-based assays, using dielectrophoresis (DEP). Cells, or mixtures of cells, are suspended in a chamber containing an array of microelectrodes located on the chamber's bottom surface. A sequence of radio frequency signals is automatically applied to the microelectrodes, and images of the DEP-induced motions of the cells are captured and analyzed to determine a characteristic parameter known as the DEP cross-over frequency. Once a cell population has been characterized in this way, the same apparatus can be used to selectively isolate cells for additional biochemical, physical, or genetic analysis. Biochemical labels or bioengineered tags such as fluorescent markers or antibody-coated beads are not required. Cell separations are achieved by “electronically tuning” into the different cell types by superimposing different signal frequencies onto the microelectrodes, using both stationary and traveling wave DEP signals. Examples of separating different types of blood cell are given, together with descriptions of cell-based bioassays that monitor physiological changes that accompany trans-membrane signaling events, apoptosis, and the aging of cell cultures.