Alireza Salmanzadeh

Investigating dielectric properties of different stages of syngeneic murine ovarian cancer cells

In this study, the electrical properties of four different stages of mouse ovarian surface epithelial (MOSE) cells were investigated using contactless dielectrophoresis (cDEP). This study expands the work from our previous report describing for the first time the crossover frequency and cell specific membrane capacitance of different stages of cancer cells that are derived from the same cell line. The specific membrane capacitance increased as the stage of malignancy advanced from 15.39 ± 1.54 mF m(-2) for a non-malignant benign stage to 26.42 ± 1.22 mF m(-2) for the most aggressive stage. These differences could be the result of morphological variations due to changes in the cytoskeleton structure, specifically the decrease of the level of actin filaments in the cytoskeleton structure of the transformed MOSE cells. Studying the electrical properties of MOSE cells provides important information as a first step to develop cancer-treatment techniques which could partially reverse the cytoskeleton disorganization of malignant cells to a morphology more similar to that of benign cells.

Dielectrophoretic differentiation of mouse ovarian surface epithelial cells, macrophages, and fibroblasts using contactless dielectrophoresis

Ovarian cancer is the leading cause of death from gynecological malignancies in women. The primary challenge is the detection of the cancer at an early stage, since this drastically increases the survival rate. In this study we investigated the dielectrophoretic responses of progressive stages of mouse ovarian surface epithelial (MOSE) cells, as well as mouse fibroblast and macrophage cell lines, utilizing contactless dielectrophoresis (cDEP). cDEP is a relatively new cell manipulation technique that has addressed some of the challenges of conventional dielectrophoretic methods. To evaluate our microfluidic device performance, we computationally studied the effects of altering various geometrical parameters, such as the size and arrangement of insulating structures, on dielectrophoretic and drag forces. We found that the trapping voltage of MOSE cells increases as the cells progress from a non-tumorigenic, benign cell to a tumorigenic, malignant phenotype. Additionally, all MOSE cells display unique behavior compared to fibroblasts and macrophages, representing normal and inflammatory cells found in the peritoneal fluid. Based on these findings, we predict that cDEP can be utilized for isolation of ovarian cancer cells from peritoneal fluid as an early cancer detection tool.

Microfluidic mixing using contactless dielectrophoresis

The first experimental evidence of mixing enhancement in a microfluidic system using contactless dielectrophoresis (cDEP) is presented in this work. Pressure‐driven flow of deionized water containing 0.5 μm beads was mixed in various chamber geometries by imposing a dielectrophoresis (DEP) force on the beads. In cDEP the electrodes are not in direct contact with the fluid sample but are instead capacitively coupled to the mixing chamber through thin dielectric barriers, which eliminates many of the problems encountered with standard DEP. Four system designs with rectangular and circular mixing chambers were fabricated in PDMS. Mixing tests were conducted for flow rates from 0.005 to 1 mL/h subject to an alternating current signal range of 0–300 V at 100–600 kHz. When the time scales of the bulk fluid motion and the DEP motion were commensurate, rapid mixing was observed. The rectangular mixing chambers were found to be more efficient than the circular chambers. This approach shows potential for mixing low diffusivity biological samples, which is a very challenging problem in laminar flows at small scales.

Multilayer contactless dielectrophoresis: Theoretical considerations

Dielectrophoresis (DEP), the movement of dielectric particles in a nonuniform electric field, is of particular interest due to its ability to manipulate particles based on their unique electrical properties. Contactless DEP (cDEP) is an extension of traditional and insulator‐based DEP topologies. The devices consist of a sample channel and fluid electrode channels filled with a highly conductive media. A thin insulating membrane between the sample channel and the fluid electrode channels serves to isolate the sample from direct contact with metal electrodes. Here we investigate, for the first time, the properties of multilayer devices in which the sample and electrode channels occupy distinct layers. Simulations are conducted using commercially available finite element software and a less computationally demanding numerical approximation is presented and validated. We show that devices can be created that achieve a similar level of electrical performance to other cDEP devices presented in the literature while increasing fluid throughput. We conclude, based on these models, that the ultimate limiting factors in device performance resides in breakdown voltage of the barrier material and the ability to generate high‐voltage, high‐frequency signals. Finally, we demonstrate trapping of MDA‐MB‐231 breast cancer cells in a prototype device at a flow rate of 1.0 mL/h when 250 VRMS at 600 kHz is applied.

Isolation of rare cancer cells from blood cells using dielectrophoresis

In this study, we investigate the application of contactless dielectrophoresis (cDEP) for isolating cancer cells from blood cells. Devices with throughput of 0.2 mL/hr (equivalent to sorting 3×106 cells per minute) were used to trap breast cancer cells while allowing blood cells through. We have shown that this technique is able to isolate cancer cells in concentration as low as 1 cancer cell per 106 hematologic cells (equivalent to 1000 cancer cells in 1 mL of blood). We achieved 96% trapping of the cancer cells at 600 kHz and 300 VRMS.

Structure, sulfatide binding properties, and inhibition of platelet aggregation by a disabled-2 protein-derived peptide.

Background: Binding of Dab2 to sulfatides results in platelet aggregation inhibition. Results: The structure of a Dab2-derived peptide (SBM) embedded in dodecylphosphocholine micelles, characterization of its minimal functional sulfatide-binding site, and its inhibitory platelet aggregation activity were determined. Conclusion: An amphipathic helical region of Dab2 SBM binds sulfatides, leading to platelet aggregation inhibition. Significance: Dab2 SBM may lead to the design of novel aggregatory inhibitors. Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for αIIbβ3 integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2-derived peptide encompassing the sulfatide-binding motifs has been determined in dodecylphosphocholine micelles using NMR spectroscopy. Dab2 sulfatide-binding motif contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes.

Disabled-2 modulates homotypic and heterotypic platelet interactions by binding to sulfatides

Disabled‐2 (Dab2) inhibits platelet aggregation by competing with fibrinogen for binding to the αIIbβ3 integrin receptor, an interaction that is modulated by Dab2 binding to sulfatides at the outer leaflet of the platelet plasma membrane. The disaggregatory function of Dab2 has been mapped to its N‐terminus phosphotyrosine‐binding (N‐PTB) domain. Our data show that the surface levels of P‐selectin, a platelet transmembrane protein known to bind sulfatides and promote cell‐cell interactions, are reduced by Dab2 N‐PTB, an event that is reversed in the presence of a mutant form of the protein that is deficient in sulfatide but not in integrin binding. Importantly, Dab2 N‐PTB, but not its sulfatide binding‐deficient form, was able to prevent sulfatide‐induced platelet aggregation when tested under haemodynamic conditions in microfluidic devices at flow rates with shear stress levels corresponding to those found in vein microcirculation. Moreover, the regulatory role of Dab2 N‐PTB extends to platelet‐leucocyte adhesion and aggregation events, suggesting a multi‐target role for Dab2 in haemostasis.

Label-free isolation and enrichment of cells through contactless dielectrophoresis.

Dielectrophoresis (DEP) is the phenomenon by which polarized particles in a non-uniform electric field undergo translational motion, and can be used to direct the motion of microparticles in a surface marker-independent manner. Traditionally, DEP devices include planar metallic electrodes patterned in the sample channel. This approach can be expensive and requires a specialized cleanroom environment. Recently, a contact-free approach called contactless dielectrophoresis (cDEP) has been developed. This method utilizes the classic principle of DEP while avoiding direct contact between electrodes and sample by patterning fluidic electrodes and a sample channel from a single polydimethylsiloxane (PDMS) substrate, and has application as a rapid microfluidic strategy designed to sort and enrich microparticles. Unique to this method is that the electric field is generated via fluidic electrode channels containing a highly conductive fluid, which are separated from the sample channel by a thin insulating barrier. Because metal electrodes do not directly contact the sample, electrolysis, electrode delamination, and sample contamination are avoided. Additionally, this enables an inexpensive and simple fabrication process. cDEP is thus well-suited for manipulating sensitive biological particles. The dielectrophoretic force acting upon the particles depends not only upon spatial gradients of the electric field generated by customizable design of the device geometry, but the intrinsic biophysical properties of the cell. As such, cDEP is a label-free technique that avoids depending upon surface-expressed molecular biomarkers that may be variably expressed within a population, while still allowing characterization, enrichment, and sorting of bioparticles. Here, we demonstrate the basics of fabrication and experimentation using cDEP. We explain the simple preparation of a cDEP chip using soft lithography techniques. We discuss the experimental procedure for characterizing crossover frequency of a particle or cell, the frequency at which the dielectrophoretic force is zero. Finally, we demonstrate the use of this technique for sorting a mixture of ovarian cancer cells and fluorescing microspheres (beads).

Investigating Dielectrophoretic Signature of Mouse Ovarian Surface Epithelial Cells, Macrophages and Fibroblasts

Epithelial ovarian carcinomas are the fourth leading cause of death in women in the United States among all cancers and the leading cause of death from gynecological malignancies1. The main reason for this high rate of mortality is the inability to properly detect these carcinomas early. Investigations for diagnosing ovarian cancer in early stages have been hindered by two major obstacles: lack of adequate cell models to study different cancer stages and lack of a reliable technique to isolate these cancer cells from peritoneal fluid. In trying to solve the first challenge, Dr. Schmelz and collaborators presented a transformed mouse ovarian surface epithelial (MOSE) cell model by isolating different transitional stages of ovarian cancer as cells progressed from a premalignant nontumorigenic phenotype to a highly aggressive malignant phenotype2, 3. In this model four stages of transformed cells, namely early (MOSE-E), early-intermediate (MOSE-E/I), intermediate (MOSE-I) and late (MOSE-L) cells, were distinguishable3. In the current study, we attempt to solve the second challenge of isolating cancer cells from macrophages and fibroblasts, which are found in the peritoneal fluid. Based on differences in cells’ intrinsic electrical properties, a new cell manipulation technique, contactless dielectrophoresis (cDEP), was implemented.Copyright

Enrichment of Cancer Cells Using a High Throughput Contactless Dielectrophoretic (CDEP) Microfluidic Device

Selective concentration of microparticles is a very important process in many medical and biological applications such as early cancer detection. It has been shown that dielectrophoresis (DEP), the motion of a particle due to its polarization in the presence of a non-uniform electric field, is a method for enrichment of bioparticels based on their electrical properties and size [1, 2, 3]. Bioparticels can be concentrated using DEP by changing applied voltage and frequency, media and particles conductivity and permittivity, and geometry of microchannel and electrodes. DEP has been used to separate circulating tumor cells (CTCs) from clinical blood, breast tumor cells from CD34+ hemopoietic stem cells, breast tumor cells from peripheral blood, leukemia cells from blood.Copyright