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.

Dielectrophoretic ultra-high-frequency characterization and in silico sorting on uptake of rare earth elements by Cupriavidus necator.

Rare earth elements (REEs) are widely used across different industries due to their exceptional magnetic and electrical properties. In this work, Cupriavidus necator is characterized using dielectrophoretic ultra-high-frequency measurements, typically in MHz range to quantify the properties of cytoplasm in C. necator for its metal uptake/bioaccumulation capacity. Cupriavidus necator, a Gram-negative bacteria strain is exposed to REEs like europium, samarium, and neodymium in this study. Dielectrophoretic crossover frequency experiments were performed on the native C. necator species pre- and post-exposure to the REEs at MHz frequency range. The net conductivity of native C. necator, Cupriavidus europium, Cupriavidus samarium, and Cupriavidus neodymium are 15.95 ± 0.029 µS/cm, 16.15 ± 0.028 µS/cm, 16.05 ± 0.029 µS/cm, 15.61 ± 0.005 µS/cm respectively. The estimated properties of the membrane published by our group are used to develop a microfluidic sorter by modeling and simulation to separate REE absorbed C. necator from the unabsorbed native C. necator species using COMSOL Multiphysics commercial software package v5.5.

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.

Dielectric Characterization and Separation Optimization of Infiltrating Ductal Adenocarcinoma via Insulator-Dielectrophoresis.

The dielectrophoretic separation of infiltrating ductal adenocarcinoma cells (ADCs) from isolated peripheral blood mononuclear cells (PBMCs) in a ~1.4 mm long Y-shaped microfluidic channel with semi-circular insulating constrictions is numerically investigated. In this work, ADCs (breast cancer cells) and PBMCs' electrophysiological properties were iteratively extracted through the fitting of a single-shell model with the frequency-conductivity data obtained from AC microwell experiments. In the numerical computation, the gradient of the electric field required to generate the necessary dielectrophoretic force within the constriction zone was provided through the application of electric potential across the whole fluidic channel. By adjusting the difference in potentials between the global inlet and outlet of the fluidic device, the minimum (effective) potential difference with the optimum particle transmission probability for ADCs was found. The radius of the semi-circular constrictions at which the effective potential difference was swept to obtain the optimum constriction size was also obtained. Independent particle discretization analysis was also conducted to underscore the accuracy of the numerical solution. The numerical results, which were obtained by the integration of fluid flow, electric current, and particle tracing module in COMSOL v5.3, reveal that PBMCs can be maximally separated from ADCs using a DC power source of 50 V. The article also discusses recirculation or wake formation behavior at high DC voltages (>100 V) even when sorting of cells are achieved. This result is the first step towards the production of a supplementary or confirmatory test device to detect early breast cancer non-invasively.

Electro-osmotic surface effects generation in an electrokinetic-based transport device: A comparison of RF and MW plasma generating sources.

It is a common practice in insulator-based dielectrophoretic separation to use and reuse PDMS-constructed microdevice for an extended period of time while performing biological and technical replicate experiments. This is usually done to rule out any effects of device variation on separation efficiency. Ensuring that all experimental conditions remain the same is critical to the conclusion that can be drawn from such repeated experiments. One important contributing factor to the flow of materials within the device is electro-osmotic velocity, which stems from the surface condition of the device construction materials. In this paper, we present an affordable microwave-based (MESA-Mgen) oxygen plasma cleaner developed for approximately less than $100 using readily obtainable parts from an average local hardware store with no specialized tools. This low-cost room-air microwave plasma generator was designed using an R-4055, 400 W, 2450 MHz half-pint household microwave oven (Sharp®) for exploring the possibility of sealing polydimethylsiloxane (PDMS) devices onto glass with minimal budgetary commitment. Microfluidic channels generated using MESA-Mgen were evaluated for their electro-osmotic velocities while factors including contact angles, storage-solvent, half-way hydrophobicity period were also explored with MESA-Mgen, and the results were compared to those obtained from the commercially available plasma cleaner (COM-PC). These outcomes revealed that the MESA-Mgen induced hydrophilicity and ensured leak-free sealing of PDMS substrates in a manner comparable with the COM-PC.

Dimethyl adenosine transferase (KsgA) contributes to cell-envelope fitness in Salmonella Enteritidis.

We previously reported that inactivation of a universally conserved dimethyl adenosine transferase (KsgA) attenuates virulence and increases sensitivity to oxidative and osmotic stress in Salmonella Enteritidis. Here, we show a role of KsgA in cell-envelope fitness as a potential mechanism underlying these phenotypes in Salmonella. We assessed structural integrity of the cell-envelope by transmission electron microscopy, permeability barrier function by determining intracellular accumulation of ethidium bromide and electrophysical properties by dielectrophoresis, an electrokinetic tool, in wild-type and ksgA knock-out mutants of S. Enteritidis. Deletion of ksgA resulted in disruption of the structural integrity, permeability barrier and distorted electrophysical properties of the cell-envelope. The cell-envelope fitness defects were alleviated by expression of wild-type KsgA (WT-ksgA) but not by its catalytically inactive form (ksgAE66A), suggesting that the dimethyl transferase activity of KsgA is important for cell-envelope fitness in S. Enteritidis. Upon expression of WT-ksgA and ksgAE66A in inherently permeable E. coli cells, the former strengthened and the latter weakened the permeability barrier, suggesting that KsgA also contributes to the cell-envelope fitness in E. coli. Lastly, expression of ksgAE66A exacerbated the cell-envelope fitness defects, resulting in impaired S. Enteritidis interactions with human intestinal epithelial cells, and human and avian phagocytes. This study shows that KsgA contributes to cell-envelope fitness and opens new avenues to modulate cell-envelopes via use of KsgA-antagonists.

Insulator-based dielectrophoretic diagnostic tool for babesiosis.

Babesia species are obligate intraerythrocytic tick-borne protozoan parasites that are the etiologic agents of babesiosis, a potentially life-threatening, malaria-like illness in humans and animals. Babesia-infected people have been known to suffer from complications including liver problems, severe hemolytic anemia, and kidney failure. As reported by the Food and Drug Administration, 38% of mortality cases observed in transfusion recipients were associated with transfusion transmitted diseases of which babesiosis is the chief culprit. As of now, no tests have been licensed yet for screening blood donors for babesiosis. Current diagnostic tools for babesiosis including enzyme-linked immunosorbent assay, fluorescence in situ hybridization, and polymerase chain reaction are expensive and burdened with multifarious shortcomings. In this research, a low-cost, high-specificity, quick, and easy-to-use insulator-based dielectrophoretic diagnostic tool is developed for characterizing and concentrating Babesia-infected cells in their homogenous mixture with healthy cell population. In this work, a mixture of Babesia-infected (varying parasitemia) and healthy red blood cells (RBCs or erythrocytes) was exposed to non-uniform electric fields in a fabricated microfluidic platform to manipulate and sort the Babesia-infected cells within a minute. At DC voltage configurations of 10 V and 0/6 V in the inlet and the two outlet channels, respectively, the diseased cells were seen to flow in a direction different from the healthy RBCs. Bright field and fluorescence microscopy were utilized to present qualitative differentiation of the healthy erythrocytes from the infected cells. The proposed micro device platform was able to enrich RBCs from 0.1% to ∼70% parasitemia. This device, when finally developed into a point-of-care diagnostic chip, would enhance the detection of Babesia-infected erythrocytes and as well serve as a precursor to babesiosis vaccine development.

Dielectrophoretic applications for disease diagnostics using lab-on-a-chip platforms.

Dielectrophoresis is a powerful technique used to distinguish distinct cellular identities in heterogeneous cell populations and to monitor changes in the cell state without the need for biochemical tags, including live and dead cells. Recent studies in the past decade have indicated that dielectrophoresis can be used to discriminate the disease state of cells by exploring the differences in the dielectric polarizabilities of the cells. Factors controlling the dielectric polarizability are dependent on the conductivity and permittivity of the cell and the suspending medium, the cell morphology, the internal structure, and the electric double layer effects associated with the charges on the cell surface. Diseased cells, such as those associated with malaria, cancer, dengue, anthrax and human African trypanosomiasis, could be spatially trapped by positive dielectrophoresis or spatially separated from other healthy cells by negative dielectrophoretic forces. The aim of this review was to provide a better and deeper understanding on how dielectrophoresis can be utilized to manipulate diseased cells. This review compiles and compares the significant findings obtained by researchers in manipulating abnormal or unhealthy cells.