Ahmet C. Sabuncu

Probing nanoparticle interactions in cell culture media.

Nanoparticle research is often performed in vitro with little emphasis on the potential role of cell culture medium. In this study, gold nanoparticle interactions with cell culture medium and two cancer cell lines (human T-cell leukemia Jurkat and human pancreatic carcinoma PANC1) were investigated. Gold nanoparticles of 10, 25, 50, and 100 nm in diameter at fixed mass concentration were tested. Size distributions and zeta potentials of gold nanoparticles suspended in deionized (DI) water and Dulbeccos Modified Eagles Media (DMEM) supplemented with fetal calf serum (FCS) were measured using dynamic light scattering (DLS) technique. In DI water, particle size distributions exhibited peaks around their nominal diameters. However, the gold nanoparticles suspended in DMEM supplemented with FCS formed complexes around 100 nm, regardless of their nominal sizes. The DLS and UV-vis spectroscopy results indicate gold nanoparticle agglomeration in DMEM that is not supplemented by FCS. The zeta potential results indicate that protein rich FCS increases the dispersion quality of gold nanoparticle suspensions through steric effects. Cellular uptake of 25 and 50 nm gold nanoparticles by Jurkat and PANC1 cell lines were investigated using inductively coupled plasma-mass spectroscopy. The intracellular gold level of PANC1 cells was higher than that of Jurkat cells, where 50 nm particles enter cells at faster rates than the 25 nm particles.

Dielectrophoretic separation of mouse melanoma clones

Dielectrophoresis (DEP) is employed to differentiate clones of mouse melanoma B16F10 cells. Five clones were tested on microelectrodes. At a specific excitation frequency, clone 1 showed a different DEP response than the other four. Growth rate, melanin content, recovery from cryopreservation, and in vitro invasive studies were performed. Clone 1 is shown to have significantly different melanin content and recovery rate from cryopreservation. This paper reports the ability of DEP to differentiate between two malignant cells of the same origin. Different DEP responses of the two clones could be linked to their melanin content.

Microfluidic impedance spectroscopy as a tool for quantitative biology and biotechnology

A microfluidic device that is able to perform dielectric spectroscopy is developed. The device consists of a measurement chamber that is 250 μm thick and 750 μm in radius. Around 1000 cells fit inside the chamber assuming average quantities for cell radius and volume fraction. This number is about 1000 folds lower than the capacity of conventional fixtures. A T-cell leukemia cell line Jurkat is tested using the microfluidic device. Measurements of deionized water and salt solutions are utilized to determine parasitic effects and geometric capacitance of the device. Physical models, including Maxwell-Wagner mixture and double shell models, are used to derive quantities for sub-cellular units. Clausius-Mossotti factor of Jurkat cells is extracted from the impedance spectrum. Effects of cellular heterogeneity are discussed and parameterized. Jurkat cells are also tested with a time domain reflectometry system for verification of the microfluidic device. Results indicate good agreement of values obtained with both techniques. The device can be used as a unique cell diagnostic tool to yield information on sub-cellular units.

Acoustophoresis in shallow microchannels

Acoustophoretic (AP) motion of spherical polystyrene particles in a steady pressure driven flow is investigated in shallow microchannels, where the channel height is comparable to the particle diameter. Particle trajectories at different ultrasonic actuation amplitudes are extracted by a particle tracking algorithm. Depths of the particles are predicted using the streamwise particle speed that is due to the pressure driven flow. The particle depths are shown to be influenced by the actuation voltage. The particle migration along the channel height is explained using the second-order perturbation theory. The particle equation of motion is employed to extract the AP force. Wall effects are included in the analysis of both particle depth and force predictions. Differences as large as 20% in the AP force magnitude due to the wall corrections are reported. The AP force is also calculated using the theoretical force expression, and compared with the experimental results. The focal length, which is the necessary distance to effectively concentrate particles in a microchannel, is calculated using the analytical solution of the particle equation of motion. The calculated force and the focusing length agree well with the experimental results. The focal length is critical for the design of micro sample concentration devices.

Dispersion state and toxicity of mwCNTs in cell culture medium with different T80 concentrations.

In this study, size distribution, zeta potential, shape, and toxicity of multi-walled carbon nanotubes (mwCNTs), and effect of non-ionic detergent Tween 80 (T80) concentrations (0%, 0.2%, and 1%) on the dispersion quality and cell viability are investigated. Nanotubes are suspended in biological solutions (DMEM, RPMI) with three different concentrations (10, 50, and 100 microg/ml) and toxicological investigations are carried on human T-cell leukemia (Jurkat) and human pancreatic carcinoma (PANC1) cell lines. According to light and transmission electron microscopy results, mwCNTs form well-defined and regular bundles in the presence of 1% T80 surfactant; whereas more irregular structures are present in absence of T80. Dispersion quality is represented in terms of the size distribution from dynamic light scattering (DLS) experiments and its second moment. Dispersion quality of the mwCNTs decreases with decreasing T80 concentration, while the constituents of RPMI and DMEM increase the dispersion quality. No significant differences between the dispersive effects of RPMI and DMEM suspensions are observed. Zeta potential of the mwCNTs is measured using electrophoretic light scattering. Variations in the nanotube and T80 concentrations do not change the zeta potential significantly. T80 concentrations above 0.2% are found to be toxic for Jurkat andPANC1 cells.

A separability parameter for dielectrophoretic cell separation.

In this study, a separability parameter is introduced to determine the selection of optimum operating parameters for DEP separation of a cell pair. The separability parameter is defined as a function of cells’ Clausius–Mossotti (CM) factors. T‐cell leukemia Jurkat and mouse melanoma B16 cells are tested to validate the separability parameter. CM factors of cells are measured using a recently developed microfluidic impedance spectroscopy device. Separability maps are generated for varying values of field frequency and buffer conductivity. Cell separation is tested using a planar interdigitated electrode array at different buffer conductivities. Impedance measurements of the DEP device are performed at various buffer conductivities. Electrode polarization effects and energy allocation for dielectrophoretic manipulation of cells are computed from the impedance data utilizing an equivalent circuit model. Cell separation results are explained in the light of the impedance measurements.

Dielectric characterization of costal cartilage chondrocytes

BACKGROUND Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy. METHODS Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes. RESULTS The results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance. CONCLUSIONS We have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues. GENERAL SIGNIFICANCE The study describes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders.

Differential Dielectric Responses of Chondrocyte and Jurkat Cells in Electromanipulation Buffers

Electromanipulation of cells as a label‐free cell manipulation and characterization tool has gained particular interest recently. However, the applicability of electromanipulation, particularly dielectrophoresis (DEP), to biological cells is limited to cells suspended in buffers containing lower amounts of salts relative to the physiological buffers. One might question the use of low conductivity buffers (LCBs) for DEP separation, as cells are stressed in buffers lacking physiological levels of salt. In LCB, cells leak ions and undergo volume regulation. Therefore, cells exhibit time‐dependent DEP response in LCB. In this work, cellular changes in LCB are assessed by dielectric spectroscopy, cell viability assay, and gene expression of chondrocytes and Jurkats. Results indicate leakage of ions from cells, increases in cytoplasmic conductivity, membrane capacitance, and conductance. Separability factor, which defines optimum conditions for DEP cell separation, for the two cell types is calculated using the cellular dielectric data. Optimum DEP separation conditions change as cellular dielectric properties evolve in LCB. Genetic analyses indicate no changes in expression of ionic channel proteins for chondrocytes suspended in LCB. Retaining cellular viability might be important during dielectrophoretic separation, especially when cells are to be biologically tested at a downstream microfluidic component.