On-Chip Impedance for Quantifying Parasitic Voltages During AC Electrokinetic Trapping.

OBJECTIVE: Assessing the effectiveness of microfluidic device structures for enabling electrokinetic or acoustic trapping requires imaging of model particles within each device under the requisite force fields. To avoid the need for extensive microscopy, the use of valuable biological samples, and reliance on a trained operator to assess efficacy of trapping, we explore electrical means to identify device geometry variations that are responsible for the poor trapping. RESULTS: Using the example of AC electrokinetic trapping over an insulated channel in a contact-less dielectrophoresis mode, we present an on-chip method to acquire impedance spectra on the microfluidic device for quantifying the parasitic voltage drops. CONCLUSION: Based on the parasitic voltage drops, device geometries can be designed to maximize fraction of the applied voltage that is available for dielectrophoretic manipulation and the measured on-chip impedance can rapidly inform downstream decisions on particle manipulation.

Characterization of sequentially-staged cancer cells using electrorotation.

The identification and separation of cells from heterogeneous populations is critical to the diagnosis of diseases. Label-free methodologies in particular have been developed to manipulate individual cells using properties such as density and morphology. The electrical properties of malignant cells, including the membrane capacitance and cytoplasmic conductivity, have been demonstrated to be altered compared to non-malignant cells of similar origin. Here, we exploit these changes to characterize individual cells in a sequentially-staged in vitro cancer model using electrorotation (EROT)-the rotation of a cell induced by a rotating electric field. Using a microfabricated device, a dielectrophoretic force to suspend cells while measuring their angular velocity resulting from an EROT force applied at frequencies between 3 kHz to 10 MHz. We experimentally determine the EROT response for cells at three stages of malignancy and analyze the resultant spectra by considering models that include the effect of the cell membrane alone (single-shell model) and the combined effect of the cell membrane and nucleus (double-shell model). We find that the cell membrane is largely responsible for a given cell's EROT response between 3 kHz and 10 MHz. Our results also indicate that membrane capacitance, membrane conductance, and cytoplasmic conductivity increase with an increasingly malignant phenotype. Our results demonstrate the potential of using electrorotation as a means making of non-invasive measurements to characterize the dielectric properties of cancer cells.

Noninvasive detection of changes in cells' cytosol conductivity by combining dielectrophoresis with optical tweezers.

Cellular electrical properties are modulated by various physical and/or chemical stresses and detection of these changes is a challenging issue. Optical tweezers (OT) and dielectrophoresis (DEP) are frequently integrated to devices dedicated to the investigation of cells properties. Here we provide a technique to detect changes in cytosol conductivity of cells by using a combination of DEP and OT. The method was exemplified for the case of cells electroporation and is based on balancing the DEP force by a controlled OT force. We observed a decrease of the DEP force in the case of electroporated cells which was correlated to a decrease of cytosol conductivity by means of Clausius-Mossotti factor modeling. For highly stressing electroporation pulses, the cytosol conductivity drops to values close to those of the cells suspending medium. These results are consistent with those reported in the literature proving the robustness of our proposed sensing method.

A feasibility study for enrichment of highly aggressive cancer subpopulations by their biophysical properties via dielectrophoresis enhanced with synergistic fluid flow.

A common problem with cancer treatment is the development of treatment resistance and tumor recurrence that result from treatments that kill most tumor cells yet leave behind aggressive cells to repopulate. Presented here is a microfluidic device that can be used to isolate tumor subpopulations to optimize treatment selection. Dielectrophoresis (DEP) is a phenomenon where particles are polarized by an electric field and move along the electric field gradient. Different cell subpopulations have different DEP responses depending on their bioelectrical phenotype, which, we hypothesize, correlate with aggressiveness. We have designed a microfluidic device in which a region containing posts locally distorts the electric field created by an AC voltage and forces cells toward the posts through DEP. This force is balanced with a simultaneous drag force from fluid motion that pulls cells away from the posts. We have shown that by adjusting the drag force, cells with aggressive phenotypes are influenced more by the DEP force and trap on posts while others flow through the chip unaffected. Utilizing single-cell trapping via cell-sized posts coupled with a drag-DEP force balance, we show that separation of similar cell subpopulations may be achieved, a result that was previously impossible with DEP alone. Separated subpopulations maintain high viability downstream, and remain in a native state, without fluorescent labeling. These cells can then be cultured to help select a therapy that kills aggressive subpopulations equally or better than the bulk of the tumor, mitigating resistance and recurrence.

Enhanced contactless dielectrophoresis enrichment and isolation platform via cell-scale microstructures.

We designed a new microfluidic device that uses pillars on the same order as the diameter of a cell (20 µm) to isolate and enrich rare cell samples from background. These cell-scale microstructures improve viability, trapping efficiency, and throughput while reducing pearl chaining. The area where cells trap on each pillar is small, such that only one or two cells trap while fluid flow carries away excess cells. We employed contactless dielectrophoresis in which a thin PDMS membrane separates the cell suspension from the electrodes, improving cell viability for off-chip collection and analysis. We compared viability and trapping efficiency of a highly aggressive Mouse Ovarian Surface Epithelial (MOSE) cell line in this 20 µm pillar device to measurements in an earlier device with the same layout but pillars of 100 µm diameter. We found that MOSE cells in the new device with 20 µm pillars had higher viability at 350 VRMS, 30 kHz, and 1.2 ml/h (control 77%, untrapped 71%, trapped 81%) than in the previous generation device (untrapped 47%, trapped 42%). The new device can trap up to 6 times more cells under the same conditions. Our new device can sort cells with a high flow rate of 2.2 ml/h and throughput of a few million cells per hour while maintaining a viable population of cells for off-chip analysis. By using the device to separate subpopulations of tumor cells while maintaining their viability at large sample sizes, this technology can be used in developing personalized treatments that target the most aggressive cancerous cells.

Dielectrophoretic field-flow fractionation of electroporated cells.

We describe the development and testing of a setup that allows for DEP field-flow fractionation (DEP-FFF) of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells based on their different polarizabilities. We first optimized the channel and electrode dimensions, flow rate, and electric field parameters for efficient DEP-FFF separation of moderately heat-treated CHO cells (50°C for 15 min) from untreated ones, with the former used as a uniform and stable model of electroporated cells. We then used CHO cells exposed to electric field pulses with amplitudes from 1200 to 2800 V/cm, yielding six groups containing various fractions of nonporated, reversibly porated, and irreversibly porated cells, testing their fractionation in the chamber. DEP-FFF at 65 kHz resulted in distinctive flow rates for nonporated and each of the porated cell groups. At lower frequencies, the efficiency of fractionation deteriorated, while at higher frequencies the separation of individual elution profiles was further improved, but at the cost of cell flow rate slowdown in all the cell groups, implying undesired transition from negative into positive DEP, where the cells are pulled toward the electrodes. Our results demonstrate that fractionation of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells is feasible at a properly selected frequency.

Dielectrophoretic field-flow microchamber for separation of biological cells based on their electrical properties.

We describe the development, fabrication and testing of a microfluidic chamber for dielectrophoretic field-flow separation of biological cells based on their electrical properties. The chamber was constructed from a single Pyrex wafer with interdigitated Au electrodes, a spacer, and a top cover glass, making the events in the chamber observable under most optical microscopes. The dimensions were optimized based on numerical computations of the electric field, its gradient and the fluid-flow velocity profile. The electrodes were fabricated using photolithography. A double-sided self-adhesive tape of 100 µm thickness was used as a spacer, with an opening of 80 mm length and 20 mm width cut in its middle to form a channel of 100 µm height, and with water-resistant acrylic glue of the tape holding the glass plates together and providing a tight seal. The glue loses its adhesive properties above 70 °C, allowing for easy disassembly of the chamber in hot water and its thorough cleaning. A 1:1 mixture of normal and 50 °C -heat-treated CHO cells was used to test the chamber. A 93% efficiency of separation was obtained, confirming the usefulness of the chamber in separating cells with sufficient differences in electrical properties of their membranes.