A novel electrophoretic assisted hydrophobic microdevice for enhancing blood cell sorting: design and numerical simulation.

Microfluidic technology has great advantages in the precise manipulation of micro-nano particles, and the hybrid microfluidic separation technology has attracted much attention due to the advantages of both active and passive separation technology at the same time. In this paper, the hydrophoresis sorting technique is combined with the dielectrophoresis technique, and a dielectrophoresis-assisted hydrophoresis microdevice is studied to separate blood cells. By using the dielectrophoresis force to change the suspension position of the cells in the channel, the scope of the hydrophoresis device for sorting particles is expanded. At the same time, the effects of microchannel width, fluid velocity, and electrode voltage on cell sorting were discussed, and the cell separation process was simulated. This work has laid a certain theoretical foundation for the rapid diagnosis of diseases in practical applications.

Probing the surface charge of condensates using microelectrophoresis.

Biomolecular condensates play an important role in cellular organization. Coacervates are commonly used models that mimic the physicochemical properties of biomolecular condensates. The surface of condensates plays a key role in governing molecular exchange between condensates, accumulation of species at the interface, and the stability of condensates against coalescence. However, most important surface properties, including the surface charge and zeta potential, remain poorly characterized and understood. The zeta potential of coacervates is often measured using laser doppler electrophoresis, which assumes a size-independent electrophoretic mobility. Here, we show that this assumption is incorrect for liquid-like condensates and present an alternative method to study the electrophoretic mobility of coacervates and in vitro condensate models by microelectrophoresis and single-particle tracking. Coacervates have a size-dependent electrophoretic mobility, originating from their fluid nature, from which a well-defined zeta potential is calculated. Interestingly, microelectrophoresis measurements reveal that polylysine chains are enriched at the surface of polylysine/polyaspartic acid complex coacervates, which causes the negatively charged protein ɑ-synuclein to adsorb and accumulate at the interface. Addition of ATP inverts the surface charge, displaces ɑ-synuclein from the surface and may help to suppress its interface-catalyzed aggregation. Together, these findings show how condensate surface charge can be measured and altered, making this microelectrophoresis platform combined with automated single-particle tracking a promising characterization technique for both biomolecular condensates and coacervate protocells.

Deformed graphene FET biosensor on textured glass coupled with dielectrophoretic trapping for ultrasensitive detection of GFAP.

Numerous efforts have been undertaken to mitigate the Debye screening effect of FET biosensors for achieving higher sensitivity. There are few reports that show sub-femtomolar detection of biomolecules by FET mechanisms but they either suffer from significant background noise or lack robust control. In this aspect, deformed/crumpled graphene has been recently deployed by other researchers for various biomolecule detection like DNA, COVID-19 spike proteins and immunity markers like IL-6 at sub-femtomolar levels. However, the chemical vapor deposition (CVD) approach for graphene fabrication suffers from various surface contamination while the transfer process induces structural defects. In this paper, an alternative fabrication methodology has been proposed where glass substrate has been initially texturized by wet chemical etching through the sacrificial layer of synthesized silver nanoparticles, obtained by annealing of thin silver films leading to solid state dewetting. Graphene has been subsequently deposited by thermal reduction technique from graphene oxide solution. The resulting deformed graphene structure exhibits higher sensor response towards glial fibrillary acidic protein (GFAP) detection with respect to flat graphene owing to the combined effect of reduced Debye screening and higher surface area for receptor immobilization. Additionally, another interesting aspect of the reported work lies in the biomolecule capture by dielectrophoretic (DEP) transport on the crests of the convex surfaces of graphene in a coplanar gated topology structure which has resulted in 10 aM and 28 aM detection limits of GFAP in buffer and undiluted plasma respectively, within 15 min of application of analyte. The detection limit in buffer is almost four decades lower than that documented for GFAP using biosensors which is is expected to pave way for advancing graphene FET based sensors towards ultrasensitive point-of-care diagnosis of GFAP, a biomarker for traumatic brain injury.

Switching Separation Migration Order by Switching Electrokinetic Regime in Electrokinetic Microsystems.

Analyte migration order is a major aspect in all migration-based analytical separations methods. Presented here is the manipulation of the migration order of microparticles in an insulator-based electrokinetic separation. Three distinct particle mixtures were studied: a binary mixture of particles with similar electrical charge and different sizes, and two tertiary mixtures of particles of distinct sizes. Each one of the particle mixtures was separated twice, the first separation was performed under low voltage (linear electrokinetic regime) and the second separation was performed under high voltage (nonlinear electrokinetic regime). Linear electrophoresis, which discriminates particles by charge, is the dominant electrokinetic effect in the linear regime; while nonlinear electrophoresis, which discriminates particles by size and shape, is the dominant electrokinetic effect in the nonlinear regime. The separation results obtained with the three particle mixtures illustrated that particle elution order can be changed by switching from the linear electrokinetic regime to the nonlinear electrokinetic regime. Also, in all cases, better separation performances in terms of separation resolution (Rs) were obtained by employing the nonlinear electrokinetic regime allowing nonlinear electrophoresis to be the discriminatory electrokinetic mechanism. These findings could be applied to analyze complex samples containing bioparticles of interest within the micron size range. This is the first report where particle elution order is altered in an iEK system.

Effect of cell shape on nonlinear electrophoresis migration.

This study contributes to the renewed interest in the study of nonlinear electrophoresis of colloidal particles. In this work the influence of cell shape on electrophoretic migration under the nonlinear regimes of moderate and strong field regimes was assessed. Four types of bacterial and yeast cells (one spherical, three non-spherical) were studied and their electrophoretic mobilities for the moderate and strong electric field magnitude regimes were estimated experimentally. The parameter of sphericity was employed to assess the effect cell shape on the nonlinear electrophoresis migration velocity and corresponding mobility under the two electric field magnitude regimes studied. As particle migration under nonlinear electrophoresis depends on particle size and shape, the results in terms of mobilities of nonlinear electrophoresis were presented as function of cell hydrodynamic diameter and sphericity. The results indicated that the magnitude of the mobilities of nonlinear electrophoresis for cells increase with increasing cell size and increase with increasing deviations from spherical shape, which is indicated by lower sphericity values. The results presented here are the very first assessment of the two types of mobilities of nonlinear electrophoresis of cells as a function of size and shape.

Preparation of Soluble Mucin Solutions from the Salivary Glands.

Studying salivary gland mucins is important for elucidating the pathogenesis of salivary gland diseases, including tumors and xerostomia, and developing diagnostic methods for them. Classic methods for isolating mucins from salivary glands require sacrificing several animals to obtain sufficient quantities of mucin and are time-consuming. Supported molecular matrix electrophoresis (SMME) was used to characterize mucins and their glycans. With this method, mucins can be analyzed within 2 days using less than 100 mg of tissue and without using expensive equipment, such as an ultracentrifuge. This chapter describes a method for preparing mucin solutions for SMME analysis of salivary gland mucins.

Supported Molecular Matrix Electrophoresis.

Distinct bands of mucins cannot be banded using a gel electrophoresis based on a molecular sieving effect due to their very large molecular weight and remarkable diversity in glycosylation. In contrast, membrane electrophoresis can separate mucins as round bands. Here, we present an analysis of mucin separation via membrane electrophoresis using a porous polyvinylidene difluoride membrane, which is highly stable against chemical modifications and various organic solvents. The separated mucins can not only be stained with dyes but also with antibodies and lectins, and glycans can be released from the excised bands and analyzed.

A facile double moving redox boundary model for visual electrophoresis titration of ascorbic acid.

In this work, we proposed a double moving redox boundary (MROB) model to realize the colorless analyte electrophoresis titration (ET) by the two steps of the redox reaction. Single MROB has been proposed for the development of ET sensing (Analyst, 2013, 138, 1137. ACS Sensor, 2019, 4, 126.), and faces great challenges in detecting the analyte without color change during redox reaction. Herein, a novel model of double-MROB electrophoresis, including its mechanisms, equations, and procedures, was developed for titration by using ascorbic acid as a model analyte. The first MROB was created with ferric iron (Fe3+) and iodide ion (I-) in which Fe3+ was reduced as Fe2+ and I- was oxidized as molecular iodine (I2) used as an indicator of visible MROB due to blue starch-iodine complex. The second boundary was then formed between the molecular iodine and model analyte of ascorbic acid. Under given conditions, there was a quantitative relationship between velocity of MROB (VMROB(ii)) and ascorbic acid concentration (CVit C) in the double-MROB system (1/VMROB(ii) = 0.6502CVit C + 4.5165, and R = 0.9939). The relevant relative standard deviation values of intraday and inter-day were less than ∼5.55% and ∼6.64%, respectively. Finally, the titration of ascorbic acid in chewable vitamin C tablets was performed by the developed method, the titration results agreed with those via the classic iodometric titration. All the results briefly demonstrated the validity of the double MROB model, in which Vit C was used as a model analyte. The developed method had potential use in quantitative analysis of redox-active species in biomedical samples.

Numerical and experimental investigation of the deviation of microparticles inside the microchannel using the vortices caused by the ICEK phenomenon.

One field of study in microfluidics is the control, trapping, and separation of microparticles suspended in fluid. Some of its applications are related to cell handling, virus detection, and so on. One of the new methods in this field is using ICEK phenomena and dielectrophoresis forces. In the present study, considering the ICEK phenomena, the microparticles inside the fluid are deviated in the desired ratio using a novel ICEK microchip. The deviation is such that after the microparticles reach the floating electrode, they are trapped in the ICEK flow vortex and deviated through a secondary channel that was placed crosswise and noncoplanar above the main channel. For simulation verification, an experimental test is done. The method used for making two noncoplanar channels and separating the particles in the desired ratio with a simple ICEK microchip is an innovation of the present study. Moreover, the adjustment of the percentage of separation of microparticles by adjusting the parameters of the applied voltage and fluid inlet velocity is one of the other innovations of the present experimental study. We observed that for input velocities of 150-1200 µm/s with applied voltages of 10-33 V, 100% of the particles can be directed toward the secondary-channel.

Accelerated Electrophoretic Focusing and Purification of DNA Based on Synchronous Coefficient of Drag Alteration.

Synchronous coefficient of drag alteration refers to a multidimensional transport mechanism where a net drift of molecules is achieved under a zero-time-average alternating motive force by perturbing their drag coefficient synchronously with the applied force. An electrophoretic form of the method is often applied to focus and purify nucleic acids in a gel under rotating electric fields. However, this method requires lengthy operation due to the use of limited field strengths. Here, using DNA as target molecules, we demonstrate that the operation time can be reduced from hours to minutes by replacing polymer gel with a microfabricated artificial sieve. We also describe an electrophoretic protocol that facilitates the collection of purified DNA from the sieve, which is shown to yield amplifiable DNA from crude samples including the lysates of cultured cells and whole blood. The sieve can be further equipped with nucleic acid amplification and detection functions for a point-of-care diagnostic application.

Using dielectrophoretic spectra to identify and separate viable yeast cells.

Immotile yeast cells were transiently moved in nonuniform sinusoidal electric fields using multiple pairs of micro-parallel cylindrical electrodes equipped with a sequential signal generator (SSG) to analyze cell viability at a clinical scale for the brewery/fermentation industry. Living yeast cells of Saccharomyces cerevisiae during the exponential-stationary phase, with a cell density of 1.15 × 105 cells mL-1 were suspended in sucrose medium. The conductivity (σs) was adjusted to 0.01 S m-1 with added KCl. Cells exposed in electric field strengths ranging from 32.89 to 40.98 kV m-1, exhibited positive dielectrophoresis (pDEP) with the lower critical frequencies (LCF) at 85.72 ± 3.59 kHz. The optimized value of LCF was shifted upwards to 780.00 ± 83.67 kHz when σswas increased to 0.10 S m-1. Dielectrophoretic and LCF spectra (translational speed of cells vs. electric field frequencies) of yeast suspensions during positive dielectrophoresis were analyzed in terms of the dielectric properties of the cell membrane and cytoplasm which reflect yeast cell viability and metabolic health status. The dielectrophoretic collection yield of cells using positive dielectrophoresis was reported on the monitor of sequential signal generator software to evaluate the number of living and dead cells through a real-time image processing analyzer. The spectra of both positive dielectrophoresis of the living and dead cells had distinguishable dielectric properties. The conductivity of the yeast cytoplasm (σc) of the dead cells was significantly less (≈ ≤ 0.05 S m-1) than that of the living yeast cells which typically had a cytoplasmic conductivity of ≈ 0.2 S m-1. This difference between viable and non-viable cells is sufficient for cell separation procedures. KEY POINTS: • Dielectrophoresis can be used to separate viable and non-viable yeast cells, • Cellular dielectric properties can be derived from the analysis of their dielectric spectra, • Cytoplasmic conductivity of viable cells is ≈ 0.2 S m-1 while that of non-viable cells ≈ ≤ 0.05 S m-1.

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.

Quantification of Free Immunoglobulin Light Chains in Urine.

BACKGROUND: The serum-free immunoglobulin light chain assay has been recommended as a screening test for monoclonal gammopathy. We evaluated the usefulness of urine free immunoglobulin light concentration for selection of specimens for immunofixation electrophoresis. METHODS: Using kits from The Binding Site for Freelite ®, we validated examination of urine for measuring free κ and λ light chains. The results of urine free light chain concentrations were evaluated to ascertain if the results could be used to reduce the number of specimens requiring urine protein immunofixation electrophoresis. RESULTS: In the 515 specimens examined, there was no evidence of monoclonal gammopathy or history of monoclonal gammopathy in 331. Monoclonal κ or λ light chains were detectable in 42 and 30 specimens, respectively. There was history of κ or λ chain associated monoclonal gammopathy in 62 and 50 patients, respectively. In the 38 monoclonal κ positive urine specimens, with light chain data, κ/λ ratio was >5.83 in all specimens. In 27 specimens positive for monoclonal λ light chains, with light chain data, the urine λ/κ ratio was > 0.17 in 24 of 27 specimens and > 0.041 in all specimens. In patients without monoclonal gammopathy all specimens had a κ/λ ratio of >5.83 or λ/κ ratio >0.17. CONCLUSIONS: The Freelite ® assay from The Binding Site is suitable for quantification of free light chains in urine. In patients with known history of monoclonal gammopathy, urine immunofixation electrophoresis may be omitted in specimens with κ/λ ratio of <5.83 for κ associated lesions and λ/κ ratio of <0.041 for λ associated lesions. However, the results do not support using this test for first-time urine testing for monoclonal light chains as it is not predictive of positive result, nor does it exclude a monoclonal light chain in urine.

Numerical modeling reveals improved organelle separation for dielectrophoretic ratchet migration.

Organelle size varies with normal and abnormal cell function. Thus, size-based particle separation techniques are key to assessing the properties of organelle subpopulations differing in size. Recently, insulator-based dielectrophoresis (iDEP) has gained significant interest as a technique to manipulate sub-micrometer-sized particles enabling the assessment of organelle subpopulations. Based on iDEP, we recently reported a ratchet device that successfully demonstrated size-based particle fractionation in combination with continuous flow sample injection. Here, we used a numerical model to optimize the performance with flow rates a factor of three higher than previously and increased the channel volume to improve throughput. We evaluated the amplitude and duration of applied low-frequency DC-biased AC potentials improving separation efficiency. A separation efficiency of nearly 0.99 was achieved with the optimization of key parameters-improved from 0.80 in previous studies (Ortiz et al. Electrophoresis, 2022;43;1283-1296)-demonstrating that fine-tuning the periodical driving forces initiating the ratchet migration under continuous flow conditions can significantly improve the fractionation of organelles of different sizes.

Low-frequency electrokinetics in a periodic pillar array for particle separation.

Deterministic Lateral Displacement (DLD) exploits periodic arrays of pillars inside microfluidic channels for high-precision sorting of micro- and nano-particles. Previously we demonstrated how DLD separation can be significantly improved by the addition of AC electrokinetic forces, increasing the tunability of the technique and expanding the range of applications. At high frequencies of the electric field (>1 kHz) the behaviour of such systems is dominated by Dielectrophoresis (DEP), whereas at low frequencies the particle behaviour is much richer and more complex. In this article, we present a detailed numerical analysis of the mechanisms governing particle motion in a DLD micropillar array in the presence of a low-frequency AC electric field. We show how a combination of Electrophoresis (EP) and Concentration-Polarisation Electroosmosis (CPEO) driven wall-particle repulsion account for the observed experimental behaviour of particles, and demonstrate how this complete model can predict conditions that lead to electrically induced deviation of particles much smaller than the critical size of the DLD array.

A review of the zone broadening contributions in free-flow electrophoresis.

The broadening of analyte streams, as they migrate through a free-flow electrophoresis (FFE) channel, often limits the resolving power of FFE separations. Under laminar flow conditions, such zonal spreading occurs due to analyte diffusion perpendicular to the direction of streamflow and variations in the lateral distance electrokinetically migrated by the analyte molecules. Although some of the factors that give rise to these contributions are inherent to the FFE method, others originate from non-idealities in the system, such as Joule heating, pressure-driven crossflows, and a difference between the electrical conductivities of the sample stream and background electrolyte. The injection process can further increase the stream width in FFE separations but normally influencing all analyte zones to an equal extent. Recently, several experimental and theoretical works have been reported that thoroughly investigate the various contributions to stream variance in an FFE device for better understanding, and potentially minimizing their magnitudes. In this review article, we carefully examine the findings from these studies and discuss areas in which more work is needed to advance our comprehension of the zone broadening contributions in FFE assays.

Light-Induced Dielectrophoresis for Characterizing the Electrical Behavior of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (hMSCs) offer a patient-derived cell source for conducting mechanistic studies of diseases or for several therapeutic applications. Understanding hMSC properties, such as their electrical behavior at various maturation stages, has become more important in recent years. Dielectrophoresis (DEP) is a method that can manipulate cells in a nonuniform electric field, through which information can be obtained about the electrical properties of the cells, such as the cell membrane capacitance and permittivity. Traditional modes of DEP use metal electrodes, such as three-dimensional electrodes, to characterize the response of cells to DEP. In this paper, we present a microfluidic device built with a photoconductive layer capable of manipulating cells through light projections that act as in situ virtual electrodes with readily conformable geometries. A protocol is presented here that demonstrates this phenomenon, called light-induced DEP (LiDEP), for characterizing hMSCs. We show that LiDEP-induced cell responses, measured as cell velocities, can be optimized by varying parameters such as the input voltage, the wavelength ranges of the light projections, and the intensity of the light source. In the future, we envision that this platform could pave the way for technologies that are label-free and perform real-time characterization of heterogeneous populations of hMSCs or other stem cell lines.

Enhancement of dielectrophoresis-based particle collection from high conducting fluids due to partial electrode insulation.

Dielectrophoresis (DEP) is a successful method to recover nanoparticles from different types of fluid. The DEP force acting on these particles is created by an electrode microarray that produces a nonuniform electric field. To apply DEP to a highly conducting biological fluid, a protective hydrogel coating over the metal electrodes is required to create a barrier between the electrode and the fluid. This protects the electrodes, reduces the electrolysis of water, and allows the electric field to penetrate into the fluid sample. We observed that the protective hydrogel layer can separate from the electrode and form a closed domed structure and that collection of 100 nm polystyrene beads increased when this occurred. To better understand this collection increase, we used COMSOL Multiphysics software to model the electric field in the presence of the dome filled with different materials ranging from low-conducting gas to high conducting phosphate-buffered saline fluids. The results suggest that as the electrical conductivity of the material inside the dome is reduced, the whole dome acts as an insulator which increases electric field intensity at the electrode edge. This increased intensity widens the high-intensity electric field factor zone resulting in increased collection. This informs how dome formation results in increased particle collection and provides insight into how the electric field can be intensified to the increase collection of particles. These results have important applications for increasing the recovery of biologically-derived nanoparticles from undiluted physiological fluids that have high conductance, including the collection of cancer-derived extracellular vesicles from plasma for liquid biopsy applications.