Insulator-based dielectrophoresis (iDEP) employs insulating structures embedded in a microchannel to produce electric field gradients. This contribution presents a detailed analysis of the regions within an iDEP system where particles are likely to be retained due to dielectrophoretic trapping in a microchannel with an array of cylindrical insulating structures. The effects of particle size and shape on dielectrophoretic trapping were analyzed by employing 1 and 2 μm polystyrene particles and Escherichia coli cells. This research aims to study the mechanism behind dielectrophoretic trapping and develop a deeper understanding of iDEP systems. Mathematical modeling with COMSOL Multiphysics was employed to assess electrokinetic and dielectrophoretic particle velocities. Experiments were carried out to determine the location of dielectrophoretic barriers that block particle motion within an iDEP microchannel; this supported the estimation of a correction factor to match experiments and simulations. Particle velocities were predicted with the model, demonstrating how the different forces acting on the particles are in equilibrium when particle trapping occurs. The results showed that particle size and shape have a significant effect on the magnitude, location, and shape of the regions of dielectrophoretic trapping of particles, which are defined by DEP isovelocity lines and EK isovelocity lines.

Current monitoring is a well-established technique for the characterization of electroosmotic (EO) flow in microfluidic devices. This method relies on monitoring the time response of the electric current when a test buffer solution is displaced by an auxiliary solution using EO flow. In this scheme, each solution has a different ionic concentration (and electric conductivity). The difference in the ionic concentration of the two solutions defines the dynamic time response of the electric current and, hence, the current signal to be measured: larger concentration differences result in larger measurable signals. A small concentration difference is needed, however, to avoid dispersion at the interface between the two solutions, which can result in undesired pressure-driven flow that conflicts with the EO flow. Additional challenges arise as the conductivity of the test solution decreases, leading to a reduced electric current signal that may be masked by noise during the measuring process, making for a difficult estimation of an accurate EO mobility. This contribution presents a new scheme for current monitoring that employs multiple channels arranged in parallel, producing an increase in the signal-to-noise ratio of the electric current to be measured and increasing the estimation accuracy. The use of this parallel approach is particularly useful in the estimation of the EO mobility in systems where low conductivity mediums are required, such as insulator based dielectrophoresis devices.

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A novel scheme for particle separation with insulator-based dielectrophoresis (iDEP) was developed. This technique offers the capability for an inverted order in particle elution, where larger particles leave the system before smaller particles. Asymmetrically shaped insulating posts, coupled with direct current (DC) biased low-frequency alternating current (AC) electric potentials, were used to successfully separate a mixture of 500 nm and 1 μm polystyrene particles (size difference of 0.5 μm in diameter). In this separation, the 1 μm particles were eluted first, demonstrating the discriminatory potential of this methodology. To extend this technique to biological samples, a mixture containing Saccharomyces cerevisiae cells (6.3 μm) and 2 μm polystyrene particles was also separated, with the cells being eluted first. The asymmetric posts featured a shorter sharp half and a longer blunt half; this produced an asymmetry in the forces exerted on the particles. The negative DC offset produced a net displacement of the smaller particles toward the upstream direction, while the post asymmetry produced a net displacement of the larger particles toward the downstream direction. This new iDEP approach provides a setup where larger particles are quickly concentrated at the outlet of the post array and can be released first when in a mixture with smaller particles. This new scheme offers an extra set of parameters (alternating current amplitude, DC offset, post asymmetry, and shape) that can be manipulated to obtain a desired separation. This asymmetric post iDEP technique has potential for separations where it is important to quickly elute and enrich larger and more fragile cells in biological samples.

Extracellular vesicles (EVs) are constantly secreted from both eukaryotic and prokaryotic cells. EVs, including those referred to as exosomes, may have an impact on cell signaling and an incidence in diseased cells. In this manuscript, a platform to capture, quantify, and phenotypically classify the EVs secreted from single cells is introduced. Microfluidic chambers of about 300 pL are employed to trap and isolate individual cells. The EVs secreted within these chambers are then captured by surface-immobilized monoclonal antibodies (mAbs), irrespective of their intracellular origin. Immunostaining against both plasma membrane and cytosolic proteins was combined with highly sensitive, multicolor total internal reflection fluorescence microscopy to characterize the immobilized vesicles. The data analysis of high-resolution images allowed the assignment of each detected EV to one of 15 unique populations and demonstrated the presence of highly heterogeneous phenotypes even at the single-cell level. The analysis also revealed that each mAb isolates phenotypically different EVs and that more vesicles were effectively immobilized when CD63 was targeted instead of CD81. Finally, we demonstrate how a heterogeneous suppression in the secreted vesicles is obtained when the enzyme neutral sphingomyelinase is inhibited.

Isolation and enrichment of low-abundant particles are essential steps in many bio-analytical and clinical applications. In this work, the capability of an insulator-based dielectrophoresis (iDEP) device for the detection and stable capture of low abundant polystyrene particles and yeast cells was evaluated. Binary and tertiary mixtures of particles and cells were tested, where the low-abundant particles had concentration ratios on the order of 1:10 000 000 compared to the other particles present in the mixture. The results demonstrated successful and stable capture and enrichment of rare particles and cells (trapping efficiencies over 99%), where particles remained trapped in a stable manner for up to 4 min. A device with four reservoirs was employed for the separation and enrichment of rare particles, where the particles of interest were first selectively concentrated and then effectively directed to a side port for future collection and analysis. The present study demonstrates that simple iDEP devices have appropriate screening capacity and can be used for handling samples containing rare particles; achieving both enrichment and isolation of low-abundant particles and cells. V C 2015 AIP Publishing LLC. [http://dx

We demonstrate the in-droplet separation and enrichment of molecules from small organic molecules to long nucleic acids (lambda DNA). Electric potentials are applied via two parallel three-dimensional electrodes, which interface the nanodroplets through polydimethylsiloxane (PDMS)–carbon composite membranes. These membranes enable the generation of uniform electric fields inside the droplets, while simultaneously preventing the formation of electrolytic byproducts. Biomolecules of different sizes migrate toward one side of the droplets, according to their net charge, when exposed to the electric field. Directly afterward, a Y-junction promotes droplet splitting, resulting in the generation of biomolecule-enriched daughter droplets. Biomolecules were fluorescently labeled, and fluorescence microscopy was employed to assess their electrophoretic separation and enrichment. Experimental results demonstrate how the enrichment of biomolecules is influenced by their size, charge, and concentration, by the ionic strength, viscosity, and pH of the suspending medium, and by the in-droplet flow profile. Enrichments above 95% were observed for small molecules and highly charged species at velocities over 10 mm/s (13 droplets per second). Moreover, the enrichment performance asymptotically approached a value of 38% for velocities as high as 50 mm/s, demonstrating the potential of this technique for the high-throughput separation of charged species. The applicability of the system was demonstrated by cleaving a peptide and selectively separating the cleaved fragments in different daughter droplets on the basis of their net charge.

The relative polarization behavior of micron and submicron polystyrene particles was investigated under direct current and very low frequency (<1 kHz) alternating current electric fields. Relative polarization of particles with respect to the suspending medium is expressed in terms of the Clausius-Mossotti factor, a parameter of crucial importance in dielectrophoretic-based operations. Particle relative polarization was studied by employing insulator-based dielectrophoretic (iDEP) devices. The effects of particle size, medium conductivity, and frequency (10-1000 Hz) of the applied electric potential on particle response were assessed through experiments and mathematical modeling with COMSOL Multiphysics(®). Particles of different sizes (100-1000 nm diameters) were introduced into iDEP devices fabricated from polydimethylsiloxane (PDMS) and their dielectrophoretic responses under direct and alternating current electric fields were recorded and analyzed in the form of images and videos. The results illustrated that particle polarizability and dielectrophoretic response depend greatly on particle size and the frequency of the electric field. Small particles tend to exhibit positive DEP at higher frequencies (200-1000 Hz), while large particles exhibit negative DEP at lower frequencies (20-200 Hz). These differences in relative polarization can be used for the design of iDEP-based separations and analysis of particle mixtures.