Dielectrophoretic traps for single-particle patterning

We present a novel microfabricated dielectrophoretic trap designed to pattern large arrays of single cells. Because flowing away untrapped cells is often the rate-limiting step during cell patterning, we designed the trap to be strong enough to hold particles against practical flow rates. We experimentally validated the trap strength by measuring the maximum flow rate that polystyrene beads could withstand while remaining trapped. These bead experiments have shown excellent agreement with our model predictions, without the use of fitting parameters. The model was able to provide us with a fundamental understanding of how the traps work, and additionally allowed us to establish a set of design rules for optimizing the traps for a wide range of cell sizes. We provide the foundations for an enabling technology that can be used to pattern cells in unique ways, allowing us to do novel cell biology experiments at the microscale.     

Experimental investigation on neural cell survival after dielectrophoretic trapping

Negative dielectrophoretic forces can effectively be used to trap cortical rat neurons. The creation of dielectrophoretic forces requires electric fields of high non-uniformity. High electric field strengths, however, can cause excessive membrane potentials by which cells may unrecoverably be changed or it may lead to cell death. In a previous study it was found that cells trapped at 3 Vtt/14 MHz did not change morphologically as compared to cells that were not exposed to the electric field. This study investigates the viability of fetal cortical rat neurons after being trapped by negative dielectrophoretic forces at frequencies up to 1 MHz. A planar quadrupole micro-electrode structure was used for the creation of a non-uniform electric field. The sinusoidal input signal was varied in amplitude (3 and 5 Vtt) and frequency (10 kHz-1 MHz). The results presented in this paper show that the viability of dielectrophoretically trapped postnatal cortical rat cells was greatly frequency dependent. To preserve viability frequencies above 100 kHz (at 3 Vtt) or 1 MHz (5 Vtt) must be used.     

Levitation and movement of human tumor cells using a printed circuit board device based on software-controlled dielectrophoresis

In this study we describe an original, efficient, and innovative printed circuit board (PCB) device able to generate dielectrophoresis-based, software-controlled cages that can be moved to any place inside a microchamber. Depending on their dielectrophoretic properties, eukaryotic cells can be "entrapped" in cages and moved under software control. The main conclusion gathered from the experimental data reported is that the PCB device based on dielectrophoresis permits levitation and movement of different tumor cells at different dielectrophoresis conditions. The results presented herein are therefore the basis for experiments aimed at forced interactions or separation of eukaryotic cells using "lab-on-a-chip." In fact, because many cages can be controlled at the same time, and two or more cages can be forced to share the same or a different location, it is possible, in principle, either to bring in contact cells of a differing histotype or to separate them.     

Introducing dielectrophoresis as a new force field for field-flow fractionation.

We present the principle of cell characterization and separation by dielectrophoretic field-flow fractionation and show preliminary experimental results. The operational device takes the form of a thin chamber in which the bottom wall supports an array of microelectrodes. By applying appropriate AC voltage signals to these electrodes, dielectrophoretic forces are generated to levitate cells suspended in the chamber and to affect their equilibrium heights. A laminar flow profile is established in the chamber so that fluid flows faster with increasing distance from the chamber walls. A cell carried in the flow stream will attain an equilibrium height, and a corresponding velocity, based on the balance of dielectrophoretic, gravitational, and hydrodynamic lift forces it experiences. We describe a theoretical model for this system and show that the cell velocity is a function of the mean fluid velocity, the voltage and frequency of the signals applied to the electrodes, and, most significantly, the cell dielectric properties. The validity of the model is demonstrated with human leukemia (HL-60) cells subjected to a parallel electrode array, and application of the device to separating HL-60 cells from peripheral blood mononuclear cells is shown.     

Dielectrophoretic registration of living cells to a microelectrode array

We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3 Ipm diameter trapping electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device.In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned approximately 20,000 cells onto aI cm2 grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70 +/- 1% fidelity versus 17 +/- 1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity.     

Dielectrophoretic registration of living cells to a microelectrode array

We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3 micromdiameter trapping electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device. In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned approximately 20000 cells onto a 1cm(2) grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70+/-1% fidelity versus 17+/-1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity.     

Dependence of dielectrophoretic force on the size of linear erythrocyte aggregates in suspension

For modeling of erythrocyte rouleaux (linear cell aggregates) we develop an approximation procedure for the dipole moment in short cylinders, which contains the case of ellipsoidal bodies as a first approximation, but allows corrections for short cylinders, more representative for such particles. In dependence on the number of erythrocytes in an aggregation, i.e., on different but discrete rouleaux lengths, the dielectrophoretic force is calculated and represented against the frequency of the applied AC field. Predictions are made for frequency regions in the 107–108 Hz range where the magnitude and the direction of dielectrophoretic forces is different for different rouleaux sizes. This property can be used for the detection and spatial separation of rouleaux populations of different length in a microelectronic array.     

An integrated laser trap/flow control video microscope for the study of single biomolecules

We have developed an integrated laser trap/flow control video microscope for mechanical manipulation of single biopolymers. The instrument is automated to maximize experimental throughput. A single-beam optical trap capable of trapping micron-scale polystyrene beads in the middle of a 200-microm-deep microchamber is used, making it possible to insert a micropipette inside this chamber to hold a second bead by suction. Together, these beads function as easily exchangeable surfaces between which macromolecules of interest can be attached. A computer-controlled flow system is used to exchange the liquid in the chamber and to establish a flow rate with high precision. The flow and the optical trap can be used to exert forces on the beads, the displacements of which can be measured either by video microscopy or by laser deflection. To test the performance of this instrument, individual biotinylated DNA molecules were assembled between two streptavidin beads, and the DNA elasticity was characterized using both laser trap and flow forces. DNA extension under varying forces was measured by video microscopy. The combination of the flow system and video microscopy is a versatile design that is particularly useful for the study of systems susceptible to laser-induced damage. This capability was demonstrated by following the translocation of transcribing RNA polymerase up to 650 s.     

Separation of polystyrene microbeads using dielectrophoretic/gravitational field-flow-fractionation.

The characterization of a dielectrophoretic/gravitational field-flow-fractionation (DEP/G-FFF) system using model polystyrene (PS) microbeads is presented. Separations of PS beads of different surface functionalization (COOH and none) and different sizes (6, 10, and 15 microm in diameter) are demonstrated. To investigate the factors influencing separation performance, particle elution times were determined as a function of particle suspension conductivity, fluid flow rate, and applied field frequency and voltage. Experimental data were analyzed using a previously reported theoretical model and good agreement between theory and experiment was found. It was shown that separation of PS beads was based on the differences in their effective dielectric properties. Particles possessing different dielectric properties were positioned at different heights in a fluid-flow profile in a thin chamber by the balance of DEP and gravitational forces, transported at different velocities under the influence of the fluid flow, and thereby separated. To explore hydrodynamic (HD) lift effects, velocities of PS beads were determined as a function of fluid flow rate in the separation chamber when no DEP field was applied. In this case, particle equilibrium height positions were governed solely by the balance of HD lift and gravitational forces. It was concluded that under the experimental conditions reported here, the DEP force was the dominant factor in controlling particle equilibrium height and that HD lift force played little role in DEP/G-FFF operation. Finally, the influence of various experimental parameters on separation performance was discussed for the optimization of DEP/G-FFF.     

Dielectrophoretic separation of monocytes from cancer cells in a microfluidic chip using electrode pitch optimization

This study proposes a microfluidic device capable of separating monocytes from a type of cancer cell that is called T-cell acute lymphoblastic leukemia (RPMI-8402) in a continuous flow using negative and positive dielectrophoretic forces. The use of both the hydrodynamic and dielectrophoretic forces allows the separation of RPMI-8402 from monocytes based on differences in their intrinsic electrical properties and sizes. The specific crossover frequencies of monocytes and RPMI-8402 cells have been obtained experimentally. The optimum ranges of electrode pitch-to-channel height ratio at the cross sections with different electrode widths have been generally calculated by numerical simulations of the gradients of the electric field intensities and calculation their effective values (root-mean-square). In the device, the cell sorting has been conducted empirically, and then, the separation performance has been evaluated by analyzing the images before and after dielectrophoretic forces applied to the cells. In this work, the design of a chip with 77 μm gold–titanium electrode pitch was investigated to achieve high purity of monocytes of 95.2%. The proposed device can be used with relatively low applied voltages, as low as 16.5 V (peak to peak). Thus, the design can be used in biomedical diagnosis and chemical analysis applications as a lab-on-chip platform. Also, it can be used for the separation of biological cells such as bacteria, RNA, DNA, and blood cells.

     

Light-induced dielectrophoretic manipulation of DNA

Light-induced dielectrophoretic movement of polystyrene beads and lambda-DNA is studied using thin films of amorphous hydrogenated silicon as local photoaddressable electrodes with a diameter of 4 microm. Positive (high-field seeking) dielectrophoretic movement is observed for both types of objects. The absence of strong negative (low-field seeking) dielectrophoresis of DNA at high frequencies is in agreement with the similarity of the dielectric constants of DNA and water, the real part of the dielectric function. The corresponding imaginary part of the dielectric function governed by the conductivity of DNA can be determined from a comparison of the frequency dependence of the dielectrophoretic drift velocity with the Clausius-Mossotti relation.     

Electrorotation and levitation of cells and colloidal particles

We review dielectrophoretic forces on cells and colloidal particles, emphasizing their use for manipulating and characterizing the electrical properties of suspended particles. Compared with dielectric spectroscopy, these methods offer a measure of independence from electrode artifacts and mixture theory. On the assumption that the particles can be modeled as uniform dielectric objects with effective dielectric properties, a simple theory can be developed for the frequency variation in the field-induced forces. For particles exhibiting counterion polarization, dielectrophoretic forces differ considerably from predictions of this theory at low frequencies, apparently because of double layer phenomena.     

Force unfolding kinetics of RNA using optical tweezers. II. Modeling experiments

By exerting mechanical force, it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles, and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article. The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that the longest handles and softest traps that allow detection of the folding/unfolding signal (handles approximately 5-10 Kbp and traps approximately 0.03 pN/nm) represent the best conditions to obtain the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules.     

The stiffness of rabbit skeletal actomyosin cross-bridges determined with an optical tweezers transducer.

Muscle contraction is brought about by the cyclical interaction of myosin with actin coupled to the breakdown of ATP. The current view of the mechanism is that the bound actomyosin complex (or "cross-bridge") produces force and movement by a change in conformation. This process is known as the "working stroke." We have measured the stiffness and working stroke of a single cross-bridge (kappa xb, dxb, respectively) with an optical tweezers transducer. Measurements were made with the "three bead" geometry devised by Finer et al. (1994), in which two beads, supported in optical traps, are used to hold an actin filament in the vicinity of a myosin molecule, which is immobilized on the surface of a third bead. The movements and forces produced by actomyosin interactions were measured by detecting the position of both trapped beads. We measured, and corrected for, series compliance in the system, which otherwise introduces large errors. First, we used video image analysis to measure the long-range, force-extension property of the actin-to-bead connection (kappa con), which is the main source of "end compliance." We found that force-extension diagrams were nonlinear and rather variable between preparations, i.e., end compliance depended not only upon the starting tension, but also upon the F-actin-bead pair used. Second, we measured kappa xb and kappa con during a single cross-bridge attachment by driving one optical tweezer with a sinusoidal oscillation while measuring the position of both beads. In this way, the bead held in the driven optical tweezer applied force to the cross-bridge, and the motion of the other bead measured cross-bridge movement. Under our experimental conditions (at approximately 2 pN of pretension), connection stiffness (kappa con) was 0.26 +/- 0.16 pN nm-1. We found that rabbit heavy meromyosin produced a working stroke of 5.5 nm, and cross-bridge stiffness (kappa xb) was 0.69 +/- 0.47 pN nm-1.     

Flexural rigidity of individual microtubules measured by a buckling force with optical traps

We used direct buckling force measurements with optical traps to determine the flexural rigidity of individual microtubules bound to polystyrene beads. To optimize the accuracy of the measurement, we used two optical traps and antibody-coated beads to manipulate each microtubule. We then applied a new analytical model assuming nonaxial buckling. Paclitaxel-stabilized microtubules were polymerized from purified tubulin, and the average microtubule rigidity was calculated as 2.0 x 10(-24) Nm2 using this novel microtubule buckling system. This value was not dependent on microtubule length. We also measured the rigidity of paclitaxel-free microtubules, and obtained the value of 7.9 x 10(-24) Nm2, which is nearly four times that measured for paclitaxel-stabilized microtubules.     

Visualizing single DNA-bound proteins using DNA as a scanning probe

Many biological processes involve enzymes moving along DNA. Such motion might be impeded by DNA-bound proteins or DNA supercoils. Current techniques are incapable of directly measuring forces that such 'roadblocks' might impose. We constructed a setup with four independently moveable optical traps, allowing us to manipulate two DNA molecules held between beads. By tightly wrapping one DNA around the other, we created a probe that can be scanned along the contour of the second DNA. We found that friction between the two polymers remains below 1 pN. Upon encountering DNA-bound proteins substantial friction forces are measured, allowing accurate localization of protein positions. Furthermore, these proteins remained associated at low probe tensions but could be driven off using forces greater than 20 pN. Finally, the full control of the orientation of two DNA molecules opens a wide range of experiments on proteins interacting with multiple DNA regions.     

Dielectrophoresis as a tool to characterize and differentiate isogenic mutants of Escherichia coli

In this study we report on an experimental method based on dielectrophoretic analysis to identify changes in four Escherichia coli isogenic strains that differed exclusively in one mutant allele. The dielectrophoretic properties of wild-type cells were compared to those of hns, hha, and hha hns mutant derivatives. The hns and hha genes code respectively for the global regulators Hha and H-NS. The Hha and H-NS proteins modulate gene expression in Escherichia coli and other Gram negative bacteria. Mutations in either hha or hns genes result in a pleiotropic phenotype. A two-shell prolate ellipsoidal model has been used to fit the experimental data, obtained from dielectrophoresis measurements, and to study the differences in the dielectric properties of the bacterial strains. The experimental results show that the mutant genotype can be predicted from the dielectrophoretic analysis of the corresponding cultures, opening the way to the development of microdevices for specific identification. Therefore, this study shows that dielectrophoresis can be a valuable tool to study bacterial populations which, although apparently homogeneous, may present phenotypic variability.     

A novel dielectrophoresis-based device for the selective retention of viable cells in cell culture media

Cost-effective production of biopharmaceuticals on a large scale can be carried out by perfusion cultures of mammalian cells. One problem with this mode of operation for submerged free-cell cultures is the requirement for an efficient cell separation device located in the effluent stream. The present work investigates the potential for the development of a novel dielectrophoresis-based cell separator, capable of providing selective retention of viable cells in cell culture media, which are highly conductive. Predictions of the dielectrophoretic (DEP) response in culture media were first obtained through a series of DEP-levitation experiments. Subsequently, a prototype microelectrode "filter" was microfabricated and tested with C174 myeloma cell suspensions of density 1 x 10(6) cells/mL. The optimum frequency range for selective retention of viable cells was found in the range 5-15 MHz. A maximum separation efficiency of 98% was achieved at 10 MHz, with an applied peak-to-peak voltage of 30 V (maximum field strength of 10(5) V/m) and a flow rate of 30 mL/h which corresponds to a superficial velocity of 5.23 cm/h through the DEP-filter channels. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 239-250, 1997.     

Modeling Complex Biological Macromolecules: Reduction of Multibead Models

The shape of simple and complex biological macromolecules can be approximated by bead modeling procedures. Such approaches are required, for example, for the analysis of the scattering and hydrodynamic behavior of the models under analysis and the prediction of their molecular properties. Using the atomic coordinates of proteins for modeling inevitably leads to models composed of a multitude of beads. In particular, for hydrodynamic modeling, a drastic reduction of the bead number may become unavoidable to enable computation. A systematic investigation of different approaches and computation modes shows that the ‘running mean’, ‘cubic grid,’ and ‘hexagonal grid’ approaches are successful, provided that the extent of reduction does not exceed a factor of 100 and the grid approaches use beads of unequal size and the beads are located at the centers of gravity. Further precautions to be taken include usage of appropriate interaction tensors for overlapping beads of unequal size and appropriate volume corrections when calculating intrinsic viscosities. The applied procedures were tested with the small protein lysozyme in a case study and were then applied to the huge capsid of the phage fr and its trimeric building block. The appearance of the models and the agreement of molecular properties and distance distribution functions of unreduced and reduced models can be used as evaluation criteria.     

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).