Research Items (35)
Microfluidic devices are widely used for biomedical applications based on microscopy or other optical detection methods. However, the materials commonly used for microfabrication typically have a high refractive index relative to water, which can create artifacts at device edges and limit applicability to applications requiring high-precision imaging or morphological feature detection. Here we present a soft lithography method to fabricate microfluidic devices out of MY133-V2000, a UV-curable, fluorinated polymer with low refractive index that is close to that of water (n = 1.33). The primary challenge in the use of this material (and fluorinated materials in general) is the low adhesion of the fluorinated material; we present several alternative fabrication methods we have tested to improve inter-layer adhesion. The close match between the refractive index of this material and aqueous solutions commonly used in biomedical applications enables fluorescence imaging at microchannel or other microfabricated edges without distortion. The close match in refractive index also enables quantitative phase microscopy imaging across the full width of microchannels without error-inducing artifacts for measurement of cell biomass. Overall, our results demonstrate the utility of low-refractive index microfluidics for biological applications requiring high-precision optical imaging.
Rapid antibody production in response to invading pathogens requires the dramatic expansion of pathogen-derived antigen-specific B lymphocyte populations. Whether B cell population dynamics are based on stochastic competition between competing cell fates, as in the development of competence by the bacteriumBacillus subtilis, or on deterministic cell fate decisions that execute a predictable program, as during the development of the wormCaenorhabditis elegans, remains unclear. Here, we developed long-term live-cell microscopy of B cell population expansion and multiscale mechanistic computational modeling to characterize the role of molecular noise in determining phenotype heterogeneity. We show that the cell lineage trees underlying B cell population dynamics are mediated by a largely predictable decision-making process where the heterogeneity of cell proliferation and death decisions at any given timepoint largely derives from nongenetic heterogeneity in the founder cells. This means that contrary to previous models, only a minority of genetically identical founder cells contribute the majority to the population response. We computationally predict and experimentally confirm nongenetic molecular determinants that are predictive of founder cells' proliferative capacity. While founder cell heterogeneity may arise from different exposure histories, we show that it may also be due to the gradual accumulation of small amounts of intrinsic noise during the lineage differentiation process of hematopoietic stem cells to mature B cells. Our finding of the largely deterministic nature of B lymphocyte responses may provide opportunities for diagnostic and therapeutic development.
- Aug 2013
Somatic cell reprogramming to pluripotency requires an immediate increase in cell proliferation and reduction in cell size. It is unknown whether proliferation and biomass controls are similarly coordinated with early events during the differentiation of pluripotent stem cells (PSCs). This impasse exists because PSCs grow in tight clusters or colonies, precluding most quantifying approaches. Here, we investigate live cell interferometry as an approach to quantify the biomass and growth of HSF1 human PSC colonies before and during retinoic acid-induced differentiation. We also provide an approach for measuring the rate and coordination of intracolony mass redistribution in HSF1 clusters using live cell interferometry images. We show that HSF1 cells grow at a consistent, exponential rate regardless of colony size and display coordinated intracolony movement that ceases with the onset of differentiation. By contrast, growth and proliferation rates show a decrease of only ∼15% decrease during early differentiation despite global changes in gene expression and previously reported changes in energy metabolism. Overall, these results suggest that cell biomass and proliferation are regulated independent of pluripotency during early differentiation, which is distinct from what occurs with successful reprogramming.
- Feb 2011
Current methods of optimizing electroosmotic (EO) pump performance include reducing pore diameter and reducing ionic strength of the pumped electrolyte. However, these approaches each increase the fraction of total ionic current carried by diffuse electric double layer (EDL) counterions. When this fraction becomes significant, concentration polarization (CP) effects become important, and traditional EO pump models are no longer valid. We here report on the first simultaneous concentration field measurements, pH visualizations, flow rate, and voltage measurements on such systems. Together, these measurements elucidate key parameters affecting EO pump performance in the CP dominated regime. Concentration field visualizations show propagating CP enrichment and depletion fronts sourced by our pump substrate and traveling at order mm/min velocities through millimeter-scale channels connected serially to our pump. The observed propagation in millimeter-scale channels is not explained by current propagating CP models. Additionally, visualizations show that CP fronts are sourced by and propagate from the electrodes of our system, and then interact with the EO pump-generated CP zones. With pH visualizations, we directly detect that electrolyte properties vary sharply across the anode enrichment front interface. Our observations lead us to hypothesize possible mechanisms for the propagation of both pump- and electrode-sourced CP zones. Lastly, our experiments show the dynamics associated with the interaction of electrode and membrane CP fronts, and we describe the effect of these phenomena on EO pump flow rates and applied voltages under galvanostatic conditions.
In this tutorial review aimed at researchers using nanofluidic devices, we summarize the current state of theoretical and experimental approaches to describing concentration polarization (CP) in hybrid microfluidic-nanofluidic systems. We also analyze experimental results for these systems and place them in the context of recent theoretical developments. We then extend the theory to explain the behavior of both positively and negatively charged, low-concentration, analyte species in systems with CP. We conclude by discussing several applications of CP in microfluidics.
- Sep 2018
The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision. © 2018, Journal of Visualized Experiments. All rights reserved.
We report the development of High Speed Live Cell Interferometry (HSLCI), a new multi-sample, multi-drug testing platform for directly measuring tumor therapy response via real-time optical cell biomass measurements. As a proof-of-concept, we show that HSLCI rapidly profiles changes in biomass in BRAF inhibitor (BRAFi) sensitive, parental melanoma cell lines and their isogenic, BRAFi-resistant sub-lines. We show reproducible results from two different HSLCI platforms at two institutions and generate biomass kinetic signatures capable of discriminating between BRAFi-sensitive and -resistant melanoma cells within 24 hours. Like other quantitative phase imaging (QPI) modalities, HSLCI is well-suited to the non-invasive measurements of single cells and cell clusters, requiring no fluorescence or dye labeling. HSLCI is substantially faster and more sensitive than field-standard growth inhibition assays and, in terms of the number of cells measured simultaneously, the number of drugs tested in parallel, and temporal measurement range, exceeds the state of the art by more than 10-fold. The accuracy and speed of HSLCI in profiling tumor cell heterogeneity and therapy resistance are promising features of potential tools to guide patient therapeutic selections.
Standard algorithms for phase unwrapping often fail for interferometric quantitative phase imaging (QPI) of biological samples due to the variable morphology of these samples and the requirement to image at low light intensities to avoid phototoxicity. We describe a new algorithm combining random walk-based image segmentation with linear discriminant analysis (LDA)-based feature detection, using assumptions about the morphology of biological samples to account for phase ambiguities when standard methods have failed. We present three versions of our method: first, a method for LDA image segmentation based on a manually compiled training dataset; second, a method using a random walker (RW) algorithm informed by the assumed properties of a biological phase image; and third, an algorithm which combines LDA-based edge detection with an efficient RW algorithm. We show that the combination of LDA plus the RW algorithm gives the best overall performance with little speed penalty compared to LDA alone, and that this algorithm can be further optimized using a genetic algorithm to yield superior performance for phase unwrapping of QPI data from biological samples.
The equal partitioning of cell mass between daughters is the usual and expected outcome of cytokinesis for self-renewing cells. However, most studies of partitioning during cell division have focused on daughter cell shape symmetry or segregation of chromosomes. Here, we use live cell interferometry (LCI) to quantify the partitioning of daughter cell mass during and following cytokinesis. We use adherent and non-adherent mouse fibroblast and mouse and human lymphocyte cell lines as models and show that, on average, mass asymmetries present at the time of cleavage furrow formation persist through cytokinesis. The addition of multiple cytoskeleton-disrupting agents leads to increased asymmetry in mass partitioning which suggests the absence of active mass partitioning mechanisms after cleavage furrow positioning.
- Nov 2014
Cell mass, volume and growth rate are tightly controlled biophysical parameters in cellular development and homeostasis, and pathological cell growth defines cancer in metazoans. The first measurements of cell mass were made in the 1950s, but only recently have advances in computer science and microfabrication spurred the rapid development of precision mass-quantifying approaches. Here we discuss available techniques for quantifying the mass of single live cells with an emphasis on relative features, capabilities and drawbacks for different applications. http://www.nature.com/cddis/journal/v5/n10/full/cddis2014420a.html
Cancer cell proliferation relies on the ability of cancer cells to grow, transition through the cell cycle, and divide. To identify novel chemical probes for dissecting the mechanisms governing cell cycle progression and cell division, and for developing new anti-cancer therapeutics, we developed and performed a novel cancer cell-based high-throughput chemical screen for cell cycle modulators. This approach identified novel G1, S, G2, and M-phase specific inhibitors with drug-like properties and diverse chemotypes likely targeting a broad array of processes. We further characterized the M-phase inhibitors and highlight the most potent M-phase inhibitor MI-181, which targets tubulin, inhibits tubulin polymerization, activates the spindle assembly checkpoint, arrests cells in mitosis, and triggers a fast apoptotic cell death. Importantly, MI-181 has broad anti-cancer activity, especially against BRAF(V600E) melanomas.
- Jan 2014
Adoptive immunotherapies against cancer, in which cytotoxic, CD8+ T cells engineered to express T cell receptors (TCRs) targeting cancer-associated antigens are transplanted into a patient, have shown dramatic promise in clinical trials. A major impediment to the widespread use of this technique for treatment of diverse cancers is the lack of a fast approach for the identification of TCRs from patient samples. In this talk, we present a method for high-throughput screening of T cell/target cell interactions by measurements of cell biomass. This live cell approach is label-free and allows cells to be recovered for downstream analysis. To ensure specificity, three parameters are tracked: target cell appearance, target cell mass loss during cell death, and T cell mass during and after the cytotoxic event (Figure panels a-c). Our results demonstrate, for the first time, the kinetics of T cell mass increase during activation. Finally, we will present an extension of this method to a microfabricated microwell format for the screening of patient samples and discovery of novel TCRs.View Large Image | View Hi-Res Image | Download PowerPoint Slide
Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20-60% over 1-4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2-3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.
Intensity images of cells on the interferometer stage after 18 h of imaging showing typical target cell conditons. Left column shows the full image frame, the right column shows a subset of the full image frame. (A)–(D) M202 target cells plated with F5 TCR transduced, CD8+ T cells showing nearly complete death of target cells. For comparison, (A) and (B) show the same field of view as in Fig. 2 A–F. (C), (D) show a single living cell. E, F. M202 target cells plated with untransduced CD8+ T cells showing viability on the stage after 18 h of imaging and cognate TCR requirement for T cell mediated cytotoxicity. (G), (H). Antigen-irrelevant PC-3 prostate cancer target cells plated with F5 TCR transduced CD8+ T cells showing the specificity of the F5 TCR. (TIF)
Four panel video showing intensity images, mass distribution images, and mass vs. time of a target M202 cell being killed by a cytotoxic T cell (CD8+, F5 TCR transduced) over the course of 5 hours of observation by LCI. (MOV)
Averaged, normalized mass versus time plots for control target cell growth conditions showing robust growth on the LCI stage, and specificity of T cell mediated cytotoxicity. (A) Unaffected M202 cells (n = 632) during treatment with F5 TCR transduced, CD8+ T cells. (B) M202 cells (n = 117) prior to treatment with F5 TCR transduced, CD8+ T cells. (C) M202 cells (n = 2058) treated with F5 TCR negative, CD8+ T cells. (D) Antigen-irrelevant, PC-3 prostate cancer cells (n = 1006) treated with F5 TCR transduced, CD8+ T cells. Blue line shows mean normalized mass versus time (normalized relative to mass at first timepoint). Light blue region shows the mean +/− SD. (TIF)
(A)–(J). Mass versus time plots for CTLs and corresponding target cells, as in Figure 4A. t = 0 h is the point at which the target cell detaches from the substrate at the beginning of cell death. CTL + target cell refers to total mass of both cells in frames where they could not be measured individually, typically due to overlap between the CTL and target cell. (TIF)
(A) Mass and (B) area histograms for activated and unresponsive T cells, relative to control experiments. Activated = activated/cytotoxic F5 TCR transduced T cells, 116 cells, n = 3 experiments. Unactivated = unactivated/unresponsive F5 TCR transduced T cells, 359 cells, n = 3 experiments. F5neg = untransduced F5 TCR negative T cells plated with M202 target cells, 530 T cells, n = 2 experiments. PC3 = F5 TCR transduced T cells plated with HLA-mismatched antigen irrelevant PC-3 prostate cancer cells, 3015 T cells, n = 3 experiments. (TIF)
Averaged, normalized mass versus time for unresponsive T cells showing steady growth on the LCI stage. (A) Unresponsive F5 TCR transduced CD8+ T cells (n = 101) plated with M202 target cells. (B) Untransduced CD8+ T cells (n = 146) plated with M202 target cells. (C) F5 TCR transduced CD8+ T cells (n = 950) plated with antigen-irrelevant, PC-3 prostate cancer target cells. (TIF)
- Jan 2013
Despite the potential high impact of human pluripotent stem cell (hPSC) research in developmental biology, cancer biology, and regenerative medicine, surprisingly little is known about how hPSCs grow, divide, and respond to their environment. In this talk, we will introduce live cell interferometery (LCI) as a new, biophysical measurement approach for precisely quantifying hPSC colony mass distributions and growth rates (Figure 1A,B,D,F). LCI is a quantitative phase microscopy technique in which the phase shift of light as it passes through and interacts with matter inside a cell is measured. Our measurements with LCI show that retinoic acid-induced differentiation minimally slows the rate of mass accumulation, a surprising result considering the large metabolic and proliferative changes associated with the transition away from the pluripotent state. We also present methods to quantify the rate and coordination of intracolony motion from colony mass distribution measurements (Figure 1 C,G). Differentiated colonies exhibit a significantly slower rate of mass motion and significantly less coordination of motion, a previously unknown behavior that may provide new information on the health and differentiation potential of available hPSC lines.View Large Image | View Hi-Res Image | Download PowerPoint Slide
Live cell mass profiling is a promising new approach for rapidly quantifying responses to therapeutic agents through picogram-scale changes in cell mass over time. A significant barrier in mass profiling is the inability of existing methods to handle pleomorphic cellular clusters and clumps, which are more commonly present in patient-derived samples or tissue cultures than are isolated single cells. Here we demonstrate automated Live Cell Interferometry (LCI) as a rapid and accurate quantifier of the sensitivity of single cell and colony-forming human breast cancer cell lines to the HER2-directed monoclonal antibody, trastuzumab (Herceptin). The relative sensitivities of small samples (<500 cells) of four breast cancer cell lines were determined tens-to-hundreds of times faster than is possible with traditional proliferation assays. These LCI advances in clustered sample assessment and speed open up the possibility for therapeutic response testing of patient-derived solid tumor samples, which are viable only for short periods ex vivo and likely to be in the form of cell aggregates and clusters.
Despite much attention to the regulation of genetic material partitioning during cell division, relatively little is known about the partitioning of cell mass, an essential outcome of successful cytokinesis. Recent work suggests that mispartitioning of cellular contents during division may constitute a form of epigenetic memory, however, conventional techniques cannot accurately quantify daughter cell masses. Quantitative phase microscopy, in which the phase shift of light as it passes through and interacts with matter inside a cell is measured, in combination with computer vision techniques, provides a new approach for directlyView Large Image | View Hi-Res Image | Download PowerPoint Slide measuring the masses of hundreds of paired daughter cells and tracking the resultant cell fates. We will show that, across several cell types, approximately one in ten cell divisions results in a highly asymmetric partitioning of mass. Additionally, we have found that specific disruption of acto-myosin activity using small molecule inhibitors leads to a marked increase in the percentage of highly-asymmetric cell divisions. This suggests that sub-cytotoxic concentrations of chemotherapeutics may lead to dramatic variation in cancer cell population mass distributions and potential epigenetic effects on cell function and disease progression.
Live Cell Interferometry (LCI) is a real time imaging technology that is extremely well suited to capture motion on the micro- and even nano-scale, with a temporal dynamic range and field of view that far exceeds scanning probe techniques. We will describe the development and application of LCI for rapid, real-time quantification of single-cell mass changes in human embryonic stem cells and populations of cells exposed to a changing external and internal environments. LCI is a conceptual advance in providing a mechanism to assess whole populations of cells, one cell at a time, for identifying, tracking/monitoring, and measuring cellular responses, such as to therapeutic drugs.View Large Image | View Hi-Res Image | Download PowerPoint Slide
- Sep 2011
A central question in cancer therapy is how individual cells within a population of tumor cells respond to drugs designed to arrest their growth. However, the absolute growth of cells, their change in physical mass, whether cancerous or physiologic, is difficult to measure directly with traditional techniques. Here, we develop live cell interferometry for rapid, real-time quantification of cell mass in cells exposed to a changing environment. We used tunicamycin induction of the unfolded protein stress response in multiple myeloma cells to generate a mass response that was temporally profiled for hundreds of cells simultaneously. Within 2 h, the treated cells were growth suppressed compared to controls, with a few cells in both populations showing a robust increase (+15%) or little change (<5%) in mass accumulation. Overall, live cell interferometry provides a conceptual advance for assessing cell populations to identify, monitor, and measure single cell responses, such as to therapeutic drugs.
- Mar 2010
We extend the analytical theory of propagating concentration polarization (CP) to describe and compare the effects of constant-voltage versus constant-current conditions on the transient development of CP enrichment and depletion zones. We support our analysis with computational and experimental results. We find that at constant voltage, enrichment and depletion regions spread as t(1/2) as opposed to the previously observed t(1) scaling for constant current conditions. At low, constant voltages, the growth and propagation of CP zones can easily be misinterpreted as nonpropagating behavior.
- Jan 2010
Porous structures with submicron pore diameters and low ionic strength electrolytes yield more efficient electroosmotic (EO) pumps. For these conditions, however, electric double layers may carry a substantial portion of ionic current, creating an imbalance between current carried by anions versus cations. This leads to the formation of net neutral regions of ion depletion and enrichment on opposite sides of the pump. We visualize ionic concentration enrichment and depletion using a custom visualization setup with an embedded porous glass EO pump. Visualizations and conductance measurements indicate that concentration polarization (CP) zones are formed and propagate in EO pump systems with order 100–1000 μM ionic strength. To date, no EO pump model has taken CP into account and yet CP has a significant effect on pumping rate and power consumption. We propose pore-volume-to-surface-area ratio as a new Duhkin number length scale for predicting CP regimes in EO pumps. CP and its propagation can have profound and long-range effects on ionic conductivity and electric fields in EO pumps.
We develop two models to describe ion transport in variable-height micro- and nanochannels. For the first model, we obtain a one-dimensional (unsteady) partial differential equation governing flow and charge transport through a shallow and wide electrokinetic channel. In this model, the effects of electric double layer (EDL) on axial transport are taken into account using exact solutions of the Poisson-Boltzmann equation. The second simpler model, which is approachable analytically, assumes that the EDLs are confined to near-wall regions. Using a characteristics analysis, we show that the latter model captures concentration polarization (CP) effects and provides useful insight into its dynamics. Two distinct CP regimes are identified: CP with propagation in which enrichment and depletion shocks propagate outward, and CP without propagation where polarization effects stay local to micro- nanochannel interfaces. The existence of each regime is found to depend on a nanochannel Dukhin number and mobility of the co-ion nondimensionalized by electroosmotic mobility. Interestingly, microchannel dimensions and axial diffusion are found to play an insignificant role in determining whether CP propagates. The steady state condition of propagating CP is shown to be controlled by channel heights, surface chemistry, and co-ion mobility instead of the reservoir condition. Both models are validated against experimental results in Part II of this two-paper series.
We present results of a combined computational and experimental study of the propagation of concentration polarization (CP) zones in a microchannel-nanochannel system. Our computational model considers the combined effects of bulk flow, electromigration, and diffusion and accurately captures the dynamics of CP. Using wall charge inside the nanochannel as a single fitting parameter, we predict experimentally observed enrichment and depletion shock velocities. Our model can also be used to compute the existence of CP with propagating enrichment and depletion shocks on the basis of measured ion mobility and wall properties. We present experiments where the background electrolyte consists of only a fluorescent ion and its counterion. These results are used to validate the computational model and to confirm predicted trends from an analytical model presented in the first of this two-paper series. We show experimentally that the enrichment region concentration is effectively independent of the applied current, while the enrichment and depletion shock velocities increase in proportion to current density.
Electroosmotic (EO) pumps can generate relatively high pressure and flow rate using no moving parts and small package volumes. Pumps with one micron (and smaller) pore diameters are promising for applications requiring high flow rate per power, such as drug delivery systems. We here show that such EO pumps exhibit significant concentration polarization (CP). CP occurs in electrokinetic devices when ionic current carried by electric double layers is significant compared to overall ionic current in the system. CP can form even in the absence of electric double layer overlap. In the CP regime, EO pump models likely fail as these models do not account for the strong spatiotemporal variations in ionic strength and associated physicochemical phenomena. We present visualizations of CP in EO pumps, and confirm that enrichment and depletion propagation front dynamics correspond with CP theory. Additionally, we show that enrichment and depletion regions are generated at and propagate from electrodes of our system, and such regions appear to interact with CP effects.
- Jan 2008
- ASME 2008 International Mechanical Engineering Congress and Exposition
Nanopores offer the potential for label-free analysis of individual proteins and low cost DNA sequencing. In order to design and evaluate nanopore devices, an understanding of nanopore electrokinetic transport is crucial. However, most studies of nanopore electrokinetic transport have neglected the effects of concentration polarization (CP) in the bulk fluid surrounding the pore. In this paper, we present a computational model which demonstrates the effects of CP on the background electrolyte in nanopore devices with tip diameters of 40–100 nm. We also present direct experimental observation of the distribution of an anionic dye in the vicinity of a conical nanopore. These results indicate that CP in a nanopore system can affect concentration distributions in the bulk solution tens of microns away from the pore, suggesting that typical boundary conditions used to model nanopore electrokinetic transport are incomplete.
- Jan 2007
- ASME 2007 International Mechanical Engineering Congress and Exposition
Recent advances in fabrication methods allow us to study and leverage the unique flow regimes offered by nano-scale fluidic channels, [1–3] and recent work suggests that the physics of microchannel/nanochannel interfaces present opportunities for novel methods of sample preconcentration and analysis. [4–6] In nanochannels, channel height is of the same order of the electric double layer (EDL) thickness, leading to a decreased electrical resistance relative to the fluidic resistance of the channel. More importantly, analyte molecules undergoing electrophoresis spend a significant amount of time within EDLs. This has a profound effect on the interfaces between micro- and nanochannels. In particular, for negatively charged walls and a nanochannel in series with two microchannels, the concentration of ions (of both signs) increases on the cathodic side of the nanochannel and decreases on the anodic side. This phenomenon is called concentration polarization (CP) or the exclusion enrichment effect. [4, 5] There is a dearth of basic studies of these phenomena and the coupling of electroosmotic flow with concentration polarization. We present experimental validation of a computational model which predicts the development of concentration polarization. Furthermore, we will show preliminary results demonstrating focusing and separation of analyte anions in the cathodic side microchannel. This focusing is due to a balance of advection and electrophoretic migration. Anionic analytes focus and separate according to electrophoretic mobility.