A continuous DC-insulator dielectrophoretic sorter of microparticles

A lab-on-a-chip device is described for continuous sorting of fluorescent polystyrene microparticles utilizing direct current insulating dielectrophoresis (DC-iDEP) at lower voltages than previously reported. Particles were sorted by combining electrokinetics and dielectrophoresis in a 250 μm wide PDMS microchannel containing a rectangular insulating obstacle and four outlet channels. The DC-iDEP particle flow behaviors were investigated with 3.18, 6.20 and 10 μm fluorescent polystyrene particles which experience negative DEP forces depending on particle size, DC electric field magnitude and medium conductivity. Due to negative DEP effects, particles are deflected into different outlet streams as they pass the region of high electric field density around the obstacle. Particles suspended in dextrose added phosphate buffer saline (PBS) at conductivities ranging from 0.50 to 8.50 mS/cm at pH 7.0 were compared at 6.85 and 17.1 V/cm. Simulations of electrokinetic and dielectrophoretic forces were conducted with COMSOL Multiphysics^® to predict particle pathlines. Experimental and simulation results show the effect of medium and voltage operating conditions on particle sorting. Further, smaller particles experience smaller iDEP forces and are more susceptible to competing nonlinear electrostatic effects, whereas larger particles experience greater iDEP forces and prefer channels 1 and 2. This work demonstrates that 6.20 and 10 μm particles can be independently sorted into specific outlet streams by tuning medium conductivity even at low operating voltages. This work is an essential step forward in employing DC-iDEP for multiparticle sorting in a continuous flow, multiple outlet lab-on-a-chip device.     

Dielectrophoretic studies of the activation of human T lymphocytes using a newly developed cell profiling system

Human T lymphocytes were stimulated using phorbol myristate acetate and ionomycin. Twenty-four hours post-activation the cells were harvested for DNA content and for measurements using a newly developed cell profiling system employing dielectrophoresis. This system provides individual cell size and dielectrophoresis data for statistically relevant numbers of control and activated cells. From this it was determined that the mean membrane specific capacitance decreased from 13.49 (+/- 4.72) mF/m(2) to 10.62 (+/- 5.13) mF/m(2). This can be related to a 21.3% reduction in the effective membrane surface area associated with membrane topography (e.g. reduction of membrane associated microvilli, blebs and folding), or to other changes of membrane architecture, following cell activation. From cytometric determinations of DNA content, it was concluded that these effects were related to a 3.0-fold decrease of cells in S-phase, and a 1.5-fold increase in G1 cells. This work demonstrates the powerful potential of using dielectrophoresis as a noninvasive tool to follow physiological changes that accompany transmembrane signaling events.     

Integrated microsystem for dielectrophoretic cell concentration and genetic detection

We have directly integrated a continuous-flow, electrokinetic method of bacterial cell concentration with room temperature, sequence-specific genetic detection. The system we have developed traps cells from a continuous-flow sample stream via dielectrophoresis, providing a 160-fold increase in cell concentration. PDMS microvalves then define a 100 nL volume around the trapped cells to which cell lysis buffer and genetic detection components are introduced. Direct, optical detection of Escherichia coli MC1061 cells is then accomplished via the sequence-specific hybridization of an rRNA-directed optical molecular beacon. This integrated microsystem is capable of sequence-specific genetic detection of 25 cells within 30 min.     

A novel ultralow conductivity electromanipulation buffer improves cell viability and enhances dielectrophoretic consistency

Cell separation has become a critical diagnostic, research, and treatment tool for personalized medicine. Despite significant advances in cell separation, most widely used applications require the use of multiple, expensive antibodies to known markers in order to identify subpopulations of cells for separation. Dielectrophoresis (DEP) provides a biophysical separation technique that can target cell subpopulations based on phenotype without labels and return native cells for downstream analysis. One challenge in employing any DEP device is the sample being separated must be transferred into an ultralow conductivity medium, which can be detrimental in retaining cells’ native phenotypes for separation. Here, we measured properties of traditional DEP reagents and determined that after just 1–2 h of exposure and subsequent culture, cells’ viability was significantly reduced below 50%. We developed and tested a novel buffer (Cyto Buffer) that achieved 6 weeks of stable shelf-life and demonstrated significantly improved viability and physiological properties. We then determined the impact of Cyto Buffer on cells’ dielectric properties and morphology and found that cells retained properties more similar to that of their native media. Finally, we vetted Cyto Buffer's usability on a cell separation platform (Cyto R1) to determine combined efficacy for cell separations. Here, more than 80% of cells from different cell lines were recovered and were determined to be >70% viable following exposure to Cyto Buffer, flow stimulation, electromanipulation, and downstream collection and growth. The developed buffer demonstrated improved opportunities for electrical cell manipulation, enrichment, and recovery for next generation cell separations.     

Immunoelectrophoresis of red blood cells performed on microcapillary chips

Immunoanalysis of blood cells on a microcapillary electrophoresis (nuCE) chip has been studied using sheep erythrocytes (ShE) as an example. Two different buffer solutions, the phosphate-buffered saline (PBS) and the gelatin veronal buffer (GVB) were examined in regard to the electrokinetic transport behavior of ShE suspended in these solutions inside the rectangular channel engraved on a quartz chip. This clarified two advantages of the use of GVB for on-chip cell electrophoresis: gelatin coatings prevent (i) nonspecific sticking of ShE on the channel wall, and cause (ii) an appreciable reduction in the zeta potential of the wall suppressing the electroosmotic flow of the buffer solution. As a result ShE suspended in the GVB can smoothly migrate from the cathode to the anode, which is the opposite flow direction of immunoglobulin G (IgG) antibodies under the physiological pH condition of 7.4. Based on these results, on-chip capillary cell immunoelectrophoresis of ShE and rabbit anti ShE antibodies (IgG) have been proposed and successfully accomplished using the GVB. It is demonstrated that the variation of the cell migration velocity originating from the change in the surface charge after binding antibodies is applicable to the fast detection of immune reactions and also to single-cell typing.     

Sample pre-concentration by isotachophoresis in microfluidic devices

We have designed microfluidic devices with the aim of coupling isotachophoresis (ITP) with zone electrophoresis (ZE) as a method to increase the concentration limit of detection in microfluidic devices. We used plastic multi-channel chips, designed with long sample injection channel segments, to increase the sample loading. The chip was designed to allow stacking of the sample into a narrow band by discontinuous ITP buffers and subsequent separation in the ZE mode. In the ITP-ZE mode, with a 2-cm long sample injection plug, sensitivity was increased by 400-fold over chip ZE and we found that the separation performance after the ITP stacking was comparable to that of regular chip ZE. We report sub-picomolar limits of detection of fluorescently labeled ACLARA eTag reporter molecules electrokinetically injected from cell lysate sample matrixes containing moderate salt concentrations. We evaluated sample injections from buffers with varied ionic strengths and found that efficient stacking and separations were obtained in both low and high conductivity buffers, including physiological buffer with at least 140 mM salt. We applied ITP-ZE to the analysis of a cell surface protease (ADAM 17) which used live intact cells in physiological buffers with detection limits below 10 cells/assay.     

Separation of malignant human breast cancer epithelial cells from healthy epithelial cells using an advanced dielectrophoresis-activated cell sorter (DACS)

In this paper, we successfully separated malignant human breast cancer epithelial cells (MCF 7) from healthy breast cells (MCF 10A) and analyzed the main parameters that influence the separation efficiency with an advanced dielectrophoresis (DEP)-activated cell sorter (DACS). Using the efficient DACS, the malignant cancer cells (MCF 7) were isolated successfully by noninvasive methods from normal cells with similar cell size distributions (MCF 10A), depending on differences between their material properties such as conductivity and permittivity, because our system was able to discern the subtle differences in the properties by generating continuously changed electrical field gradients. In order to evaluate the separation performance without considering size variations, the cells collected from each outlet were divided into size-dependent groups and counted statistically. Following that, the quantitative relative ratio of numbers between MCF 7 and MCF 10A cells in each size-dependent group separated by the DEP were compared according to applied frequencies in the range 48, 51, and 53 MHz with an applied amplitude of 8 V_pp. Finally, under the applied voltage of 48 MHz–8 V_pp and a flow rate of 290 μm/s, MCF 7 and MCF 10A cells were separated with a maximum efficiency of 86.67% and 98.73% respectively. Therefore, our suggested system shows it can be used for detection and separation of cancerous epithelial cells from noncancerous cells in clinical applications.     

Electrokinetic sample transport in a microchannel with spatial electrical conductivity gradients

Studies of the sample transport in a microchannel with the electrical conductivity gradient are critical to develop techniques for on-chip sample transport control. A numerical model presented in this paper, consisting of the electrical potential equation, full Navier-Stokes equation and species conservation equation, is used to simulate sample transport in a microchannel with the consideration of the conductivity gradient. There are two situations studied here, sample pumping (where sample separation is minimized by employing a high-conductivity buffer in the sample region), and sample stacking (where sample separation is expedited by using a low-conductivity buffer as the sample carrier). The effects of applied electrical potential, sample diffusion coefficient and the ratio of conductivity of the driving buffer over the sample carrying buffer are investigated by using the developed model.     

Thousandfold signal increase using field-amplified sample stacking for on-chip electrophoresis

Field-amplified sample stacking (FASS) leverages conductivity gradients between a volume of injected sample and the background buffer to increase sample concentration. A major challenge in applying FASS to on-chip assays is the initial setup of high-conductivity gradient boundaries in the region of the injected sample volume. We have designed, fabricated, and characterized a novel FASS-capillary electrophoresis (CE) chip design that uses a photoinitiated porous polymer structure to facilitate sample injection and flow control for high-gradient FASS. This polymer structure provides a region of high flow resistance that allows the electromigration of sample ions. We have demonstrated an electropherogram signal increase by a factor of 1100 in electrophoretic separations of fluorescein and Bodipy with, respectively, 2 microM and 1 microM initial concentrations.     

Electrophoretic separations of neuromediators on microfluidic devices

In the present work, on-chip capillary electrophoresis for the separation of neuromediators is demonstrated. The influence of separation buffer (composition, pH, SDS additive), on-chip electrokinetic sample stacking, and surface pretreatment of the PDMS-PDMS and hybrid PDMS-glass devices on the electrokinetic characteristics of microfluidics (ν_eo, μ_eo, ζ) and separation performance of on-chip capillary electrophoresis of neuromediators have been investigated. It is demonstrated that for the effective separation of neuropeptides on elastomer-based microfluidic devices, on-chip sample stacking is necessary. Field-amplified sample stacking for electroosmotic flow supported on-chip separations of neuromediators and without special design of the sample injection scheme has been demonstrated. Electrophoretic separations of fluorescently labeled analytes have been achieved within tens of seconds at injection volumes of about 110 pL, with plate numbers varying from <1000 to ∼22,000. These results demonstrate that on-chip separation methods with hybrid PDMS-glass devices are perspective for the analysis of (neuro)peptides in small volumes.     

Development of an on-chip injector for microchip-based flow analyses using laminar flow

A new on-chip injector for microchip-based flow analyses has been designed and characterized. The microchip design utilizes separate laminar flow streams of buffer and sample that are brought into parallel contact for a distance of 300 microm. The buffer flow stream is first routed through a conventional 6-port injection valve fitted with a 5 microm i.d. sample loop. When the 6-port valve is actuated from load to inject for a given time, the on-chip buffer flow stream is constricted and the sample flow stream is pressurized into the buffer flow channel. Once the valve returns to the load state the separate laminar flow streams resume. Fluorescence detection was used to characterize the injector and it was found that 50 injections of a 100 microM fluorescein sample led to an average peak height of 174.32 +/- 2.05 AFU (RSD 1.18%) and average peak skew of 1.37 +/- 0.06. The injector was also interfaced with amperometric detection. Injections of catechol solutions ranging in concentration from 500 nM to 100 microM resulted in a linear response (sensitivity = 2.49 pA microM(-1), r(2) = 0.998) and a limit of detection of 155 nM (S/N = 3). Compared to an off-chip injection scheme, plug dilution, band broadening, and peak asymmetry are much reduced. Finally, the injection and subsequent lysis of an erythrocyte sample was demonstrated, with an injected plug of erythrocytes being lysed 5.72 +/- 0.15 s after injection into a flow stream containing sodium dodecyl sulfate (n = 10). The new injection scheme does not require complex valving mechanisms or high pressures and enables reproducible injections from a continuous sample flow stream in a manner where changes in analyte concentration can be monitored with high temporal resolution.     

Effects of liquid conductivity differences on multi-component sample injection, pumping and stacking in microfluidic chips

As an increasing number of processes are being integrated into Lab-on-a-chip devices, there is an increasing need for flexible and accurate sample manipulation techniques for effective transport and separation. Conductivity differences between running buffer and analyte samples can arise as a product of on-chip processing, or by design. The two situations studied here are sample pumping (where bulk transport is increased and separation of charged analytes is delayed using a relatively high conductivity sample), and sample stacking (where bulk transport is decreased and separation of charged analytes is expedited using a relatively low conductivity sample). A recently developed dynamic loading method for on-chip sample injection in a straight-cross channel configuration is applied here to both pumping and stacking cases. A key characteristic of the dynamic loading method is the ability to inject samples of high concentration density and uniformity of any length. By employing the conductivity differences alone, the effectiveness of either sample transport or sample separation are shown to improve over the uniform conductivity case. Then it is demonstrated that increasing the sample length, through dynamic loading, greatly increases the effectiveness of sample pumping, evidenced in an eight-fold increase in peak height as well as a decrease in total sample length at a downstream detector. Dynamic loading in the sample stacking case was shown to also increase peak intensity height (three-fold) in rapid separations. These results demonstrate that the dynamic loading technique, used in conjunction with strategic conductivity differences, significantly extends the capabilities of microfluidic chips.     

Chemical responses of single yeast cells studied by fluorescence microspectroscopy under solution-flow conditions

A microspectroscopy system combined with a fluid manifold was developed to manipulate and analyze "single" living cells. A sample buffer solution containing living cells was introduced into a flow cell set on a thermostated microscope stage and a few cells were allowed to attach to the bottom wall of the flow cell. With these living cells being attached to the wall, other floating cells were pumped out by flowing a buffer solution. These procedures made it possible to keep a few cells in the flow cell and to analyze single cells by fluorescence microspectroscopy. The technique was applied to study the time course of staining processes of single living yeast (Saccharomyces cerevisiae) cells by using two types of a fluorescent probe. The present methodology was shown to be of primary importance for obtaining biochemical/physiological information on single living cells and also for studying cell-to-cell variations in several characteristics.     

Direct current insulator-based dielectrophoretic characterization of erythrocytes: ABO-Rh human blood typing

A microfluidic platform developed for quantifying the dependence of erythrocyte (red blood cell, RBC) responses by ABO-Rh blood type via direct current insulator dielectrophoresis (DC-iDEP) is presented. The PDMS DC-iDEP device utilized a 400 x 170?μm2 rectangular insulating obstacle embedded in a 1.46-cm long, 200-μm wide inlet channel to create spatial non-uniformities in direct current (DC) electric field density realized by separation into four outlet channels. The DC-iDEP flow behaviors were investigated for all eight blood types (A+, A-, B+, B-, AB+, AB-, O+, O-) in the human ABO-Rh blood typing system. Three independent donors of each blood type, same donor reproducibility, different conductivity buffers (0.52-9.1?mS/cm), and DC electric fields (17.1-68.5?V/cm) were tested to investigate separation dependencies. The data analysis was conducted from image intensity profiles across inlet and outlet channels in the device. Individual channel fractions suggest that the dielectrophoretic force experienced by the cells is dependent on erythrocyte antigen expression. Two different statistical analysis methods were conducted to determine how distinguishable a single blood type was from the others. Results indicate that channel fraction distributions differ by ABO-Rh blood types suggesting that antigens present on the erythrocyte membrane polarize differently in DC-iDEP fields. Under optimized conductivity and field conditions, certain blind blood samples could be sorted with low misclassification rates.     

Continuous dielectrophoretic bacterial separation and concentration from physiological media of high conductivity

Biological sample processing involves purifying target analytes from various sample matrices and concentrating them to a small volume from a large volume of crude sample. This complex process is the major obstacle for developing a microfluidic diagnostic platform. In this study, we present a microfluidic device that can continuously separate and concentrate pathogenic bacterial cells from complex sample matrices such as cerebrospinal fluid and whole blood. Having overcome critical limitations of dielectrophoretic (DEP) operation in physiological media of high conductivity, we utilized target specific DEP techniques to incorporate cell separation, medium exchange, and target concentration into an integrated platform. The proposed microfluidic device can uptake mL volumes of crude biological sample and selectively concentrate target cells into a submicrolitre volume, providing ~10(4) fold of concentration. We designed the device based on the electrokinetic theory and electric field simulation, and tested the device performance with different sample types. The separation efficiency of the device was as high as 97.0% for a bead mixture in TAE buffer and 94.3% and 87.2% for E. coli in human cerebrospinal fluid and blood, respectively. A capture efficiency of 100% was achieved in the concentration chamber. With a relatively simple configuration, the proposed device provides a robust method of continuous sample processing, which can be readily integrated into a fully automated microfluidic diagnostic platform for pathogen detection and quantification.     

Feasibility study for cell electroporation detection and separation by means of dielectrophoresis

Electroporation is a phenomenon during which exposure of a cell to high voltage electric pulses results in a significant increase in its membrane permeability. Aside from the fact that after the electroporation the cell membrane becomes more permeable, the cells' geometrical and electrical properties change considerably. These changes enable use of the force on dielectric particles exposed to non-uniform electric field (dielectrophoresis) for separation of non-electroporated and electroporated cells. This paper reports the results of an attempt to separate non-electroporated and electroporated cells by means of dielectrophoresis. In several experiments we managed to separate the non-electroporated and electroporated cells suspended in a medium with conductivity 0.174 S/m by exposing them to a non-uniform electric field at a frequency of 2 MHz. The behaviour of electroporated cells exposed to dielectrophoresis raises the presumption that in addition to conductivity, considerable changes in membrane permittivity occur after the electroporation.     

Dielectrophoretic sorting on a microfabricated flow cytometer: label free separation of Babesia bovis infected erythrocytes

Dielectrophoresis is a method that has demonstrated great potential in cell discrimination and isolation. In this study, the dielectrophoretic sorting of normal and Babesia bovis infected erythrocytes was performed using a microfabricated flow cytometer. Separation was possible through exploitation of the dielectric differences between normal and infected erythrocytes, essentially due to the higher ionic membrane permeability of B. bovis infected cells. Sorting experiments were performed inside a microchip made from Pt microelectrodes and SU-8 channels patterned on a glass substrate. Optimum cell separation was achieved at 4 MHz using an in vitro culture of B. bovis suspended in 63 mS/m phosphate buffer and applying a sinusoidal voltage of 15 V peak-to-peak. Normal erythrocytes experienced stronger positive dielectrophoresis (pDEP) than B. bovis infected cells, moving them closer to the microelectrodes. Under these conditions it was possible to enrich the fraction of infected cells from 7 to 50% without the need of extensive sample preparation or labelling. Throughout the experiments very few microliters of sample were used, suggesting that this system may be considered suitable for integration in a low-cost automated device to be used in the in situ diagnostic of babesiosis.     

Insulator-based dielectrophoretic single particle and single cancer cell trapping

Trapping of individual cells at specific locations in a microfluidic lab-on-a-chip platform is essential for single cell studies, especially those requiring individual stimulation followed by downstream analysis. To this aim, we have designed microdevices based on direct current (DC) insulator-based dielectrophoresis (iDEP) acting as individual single cell traps. We present both the design of a negative iDEP trap and a positive iDEP trap using insulating posts integrated at microchannel intersections. We obtained electric field distributions via numerical simulations adapted to the intersection and trap geometry with which we predict single particle pathlines. With polystyrene particles of 10?μm diameter, we demonstrated an effective design for a single particle trap in the case of negative dielectrophoresis. The onset trapping voltage shows an inverse relation to the buffer conductivity, thus indicating the influence of electrokinetic effects on the trapping behavior. Additionally, we demonstrated the proof-of-principle of single MCF-7 breast cancer cell trapping in a positive iDEP trap. Our single particle trapping experiments were further in very good agreement with numerical simulations. To ensure that no significant damage occurred to the cells during the experiment, we further optimized medium conditions to ensure viability of the cells for at least 1?h, more than sufficient for microfluidic trapping experiments. Our results thus indicated the successful design of DC iDEP traps, which can easily be integrated into a variety of microchip operations for single cell analysis.     

Bovine red blood cell starvation age discrimination through a glutaraldehyde-amplified dielectrophoretic approach with buffer selection and membrane cross-linking

We report a novel buffer electric and dielectric relaxation time tuning technique, coupled with a glutaraldehyde (Glt.) cross-linking cell fixation reaction that allows for sensitive dielectrophoretic analysis and discrimination of bovine red blood cells of different starvation age. Guided by a single-shell oblate spheroid model, a zwitterion buffer composition is selected to ensure that two measurable crossover frequencies (cof's) near 500 kHz exist for dielectrophoresis (DEP) within a small range of each other. It is shown that the low cof is sensitive to changes in the cell membrane dielectric constant, in which cross-linking by Glt. reduces the dielectric constant of the cell membrane from 10.5 to 3.8, while the high cof is sensitive to cell cytoplasm conductivity changes. We speculate that this enhanced particle polarizability that results from the cross-linking reaction is because younger (reduced starvation time) cells possess more amino groups that the reaction can release to enhance the cell interior ionic strength. Such sensitive discrimination of cells with different age (surface protein density) by DEP is not possible without the zwitterion buffer and cleavage by Glt. treatment. It is then expected that rapid identification and sorting of healthy from diseased cells can be similarly sensitized.     

Temperature measurements in microfluidic systems: heat dissipation of negative dielectrophoresis barriers

The manipulation of living biological cells in microfluidic channels by a combination of negative dielectrophoretic barriers and pressure-driven flows is widely employed in lab-on-a-chip systems. However, electric fields in conducting media induce Joule heating. This study investigates if the local temperatures reached under typical experimental conditions in miniaturized systems cause a potential risk for hyperthermic stress or cell damage. Two methods of optical in situ temperature detection have been tested and compared: (i) the exposure of the thermo-dependent fluorescent dye Rhodamine B to heat sources situated in microfluidic channels, and (ii) the use of thermoprecipitating N-alkyl-substituted acrylamide polymers as temperature threshold probes. Two-dimensional images of temperature distributions in the vicinity of active negative dielectrophoresis (nDEP)-barriers have been obtained and local temperature variations of more than 20 degrees C have been observed at the electrode edges. Heat propagation via both buffer and channel walls lead to significant temperature increases within a perimeter of 100 microm and more. These data indicate that power dissipation has to be taken into account when experiments at physiological temperatures are planned.