High-throughput dynamical analysis of dielectrophoretic frequency dispersion of single cells based on deflected flow streamlines

Abstract

Phenotypic quantification of cells based on their plasma membrane capacitance and cytoplasmic conductivity, as determined by their dielectrophoretic frequency dispersion, is often used as a marker for their biological function. However, due to the prevalence of phenotypic heterogeneity in many biological systems of interest, there is a need for methods capable of determining the dielectrophoretic dispersion of single cells at high throughput and without the need for sample dilution. We present a microfluidic device methodology wherein localized constrictions in the microchannel are used to enhance the field delivered by adjoining planar electrodes, so that the dielectrophoresis level and direction on flow-focused cells can be determined on each traversing cell in a high-throughput manner based on their deflected flow streamlines. Using a sample of human red blood cells diluted to 2.25 × 10⁸ cells/mL, the dielectrophoretic translation of single cells traversing at a flow rate of 1.68 μL/min is measured at a throughput of 1.1 × 10⁵ cells/min, to distinguish positive versus negative dielectrophoresis and determine their crossover frequency in media of differing conductivity for validation of the computed membrane capacitance to that from prior methods. We envision application of this dynamic dielectrophoresis (Dy-DEP) method towards high-throughput measurement of the dielectric dispersion of single cells to stratify phenotypic heterogeneity of a particular sample based on their DEP crossover frequency, without the need for significant sample dilution. Grapical abstract.

Keywords: Cytometry; Dielectrophoresis; Membrane capacitance; Microfluidics; Phenotype.

Figure 1.

Schematic of microfluidic device for dynamic dielectrophoresis (Dy-DEP): (a) Functioning principle based on balance of nDEP versus drag forces; (b) overall chip design; (c) focusing effect of the sheath flow pushes cells in the sample away from electrodes and towards the constriction regions of the device; (d) example differences in fluid flow streamlines of cell types with differing DEP response.

Figure 2.

2D simulations of the electric field (V/m) profiles for: (a) constriction channel of Dy-DEP design versus (c) channel design with electrodes only. The field profiles for the two devices across the probe-lines per (b) horizontal probe-lines (top), vertical probe-lines (bottom) are shown in (d) for E-field norm variation in the x-coordinate for the 3D constriction channel: A-A’, B-B’, C-C’ versus on the equivalent straight channel with electrodes: a-a’, b-b’, c-c’. (e) E-field norm variation in the y-coordinate, per the inset for the E-field variation on the tip (C-C’).

Figure 3.

3D simulations of the Electric field norm (V/m) distribution in: (a) Dy-DEP design with 3D constrictions coupled to planar electrodes (see inset for 3D E-field distribution between the planar electrodes and the constriction tip) compared to (b) the equivalent straight channel design with 3D electrodes. The colors are adjusted for equivalent field levels to present the relative differences in field extent, but (a) extends to a higher level of maximum field versus (b).

Figure 4.

Particle tracing simulations with model cell types for optimizing design and operating conditions for the separation of RBCs (red of 5 μm) versus platelets (blue of 1.8 μm). (a) No applied Voltage (no DEP) causes the undeflected cells to be scattered at the outlet (right inset). (b) Applied Voltage (50 V_pp) shows significantly higher nDEP deflection of RBCs versus platelets (at 100 kHz with a media conductivity of 550 μS/cm), causing spatial separation in their flow streamlines (per inset).

Figure 5.

Effect of dielectrophoretic translation on flow trajectories of human red blood cells (hRBCs) at a sample concentration of 2.25 × 10⁸ cells/mL at a total flow rate of 1.68 μL/min (sample flow of 0.48 μL/min plus focusing sheath flow of 1.2 μL/min) for measurement at a throughput of 1.1x10⁵ cells/min.: (a) No applied voltage. (b-j) with applied voltages of ~60 V_pp across 150 μm spaced electrodes at indicated media conductivities (vertical axis) and frequencies (horizontal axis), with the DEP level and direction indicated by labels.

Figure 6.

Intensity threshold plots obtained from phase contrast microscopy images are used to assess the ability to discern differences in dielectrophoresis level and direction based on the flow streamlines: (a) summary data box plot with range of histograms in displaced position of traversing RBCs from edge of channel wall (y-direction) under the conditions from Fig 5, including: (b) pDEP versus nDEP deflection is clearly distinguished based on lateral separations in streamlines of > 20 μm at 154 μS/cm and ~40 μm comparing pDEP at 17 μS/cm to nDEP at 154 μS/cm (see Fig. 6a); (c) strong pDEP causes focusing of RBCs to within 15 μm of wall edge versus the highly dispersed profile under no DEP; (d) weak nDEP focuses RBCs at least 60 μm away from wall edge, and (e) weak pDEP focuses RBCs to within 30 μm off the wall edge, in comparison to the highly dispersed profile under no DEP; (f) strong nDEP is also distinguished well versus no DEP behavior. For comparison, the displacement range for the FIELD OFF condition is also indicated as an arrow in (a) (95% confidence level).