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In the growing field of capacitive deionization (CDI), a number of performance metrics have emerged to describe the desalination process. Unfortunately, the separation conditions under which these metrics are measured are often not specified, resulting in optimal performance at minimal removal. Here we outline a system of performance metrics and reporting conditions that resolves this issue. Our proposed system is based on volumetric energy consumption (Wh/m3) and throughput productivity (L/h/m2) reported for a specific average concentration reduction, water recovery, and feed salinity. To facilitate and rationalize comparisons between devices, materials, and operation modes, we propose a nominal standard separation of removing 5 mM from a 20 mM NaCl feed solution at 50% water recovery. We propose this particular separation as a standard, but emphasize that the rationale presented here applies irrespective of separation details. Using our proposed separation, we compare the desalination performance of a flow-through electrode (fte-CDI) cell and a flow between membrane (fb-MCDI) device, showing how significantly different systems can be compared in terms of generally desirable desalination characteristics. In general, we find that performance analysis must be considered carefully so to not allow for ambiguous separation conditions or the maximization of one metric at the expense of another. Additionally, for context and clarity, we discuss a number of important underlying performance indicators and cell characteristics that are not performance measures in and of themselves but can be examined to better understand differences in performance.

Efficient water removal from a cool surface during condensation is critically important to the enhancement of a variety of heat transfer applications. Previous work has focused on the fabrication of superhydrophobic surfaces which promote water droplets and removal via droplet shedding or jumping. Here, we report a novel strategy with a droplet self-cleaning surface which spontaneously transports all of the droplets from the condensation surface to the back side. We fabricate the self-cleaning surface by simply tailoring the wettability of the two sides of a porous membrane and demonstrate that the hydrophobic side is effective in clearing off droplets of a wide range of diameters. Even during rapid impingement of droplets smaller than 10 μm, this surface remains dry. We further demonstrate a "dry condensation" process wherein a surface undergoing rapid condensation is maintained free of droplets. This minimizes the essential thermal resistance of the process, and we estimate a twofold increase in condensation rate compared with a simple copper surface under the same conditions. Our method is tailorable, extendable to a wide range of materials and geometries, and shows great potential for a broad range of condensation processes.

In this work, we consider a microfluidic mixer that uses hydrodynamic diffusion stream to induce the beginning of the folding process of a certain protein. To perform these molecular changes, the concentration of the denaturant, which is introduced into the mixer together with the protein, has to be diminished until a given value in a short period of time, known as mixing time. In this context, this article is devoted to optimize the design of the mixer, focusing on its shape and its flow parameters with the aim of minimizing its mixing time. First, we describe the involved physical phenomena through a mathematical model that allows us to obtain the mixing time for a considered device. Then, we formulate an optimization problem considering the mixing time as the objective function and detailing the design parameters related to the shape and the flow of the mixer. For dealing with this problem, we propose an enhanced optimization algorithm based on the hybridization of two techniques: a genetic algorithm as a core method and a multi-layer line search methodology based on the secant, which aims to improve the initialization of the core method. More precisely, in our hybrid approach, the core optimization is implemented as a sub-problem to be solved at each iteration of the multi-layer algorithm starting from the initial conditions that it provides. Before applying it to the mixer design problem, we validate this methodology by considering a set of benchmark problems and, then, compare its results to those obtained with other classical global optimization methods. As shown in the comparison, for the majority of those problems, our methodology needs fewer evaluations of the objective function, has higher success rates and is more accurate than the other considered algorithms. For those reasons, it has been selected for solving the computationally expensive problem of optimizing the mixer design. The obtained optimized device shows a great reduction in its mixing time with respect to the state-of-the-art mixers.

Capacitive deionization (CDI) performance metrics can vary widely with operating methods. Conventional CDI operating methods such as constant current and constant voltage show advantages in either energy or salt removal performance, but not both. We here develop a theory around and experimentally demonstrate a new operation for CDI that uses sinusoidal forcing voltage (or sinusoidal current). We use a dynamic system modeling approach, and quantify the frequency response (amplitude and phase) of CDI effluent concentration. Using a wide range of operating conditions, we demonstrate that CDI can be modeled as a linear time invariant system. We validate this model with experiments, and show that a sinusoid voltage operation can simultaneously achieve high salt removal and strong energy performance, thus very likely making it superior to other conventional operating methods. Based on the underlying coupled phenomena of electrical charge (and ionic) transfer with bulk advection in CDI, we derive and validate experimentally the concept of using sinusoidal voltage forcing functions to achieve resonance-type operation for CDI. Despite the complexities of the system, we find a simple relation for the resonant time scale: the resonant time period (frequency) is proportional (inversely proportional) to the geometric mean of the flow residence time and the electrical (RC) charging time. Operation at resonance implies the optimal balance between absolute amount of salt removed (in moles) and dilution (depending on the feed volume processed), thus resulting in the maximum average concentration reduction for the desalinated water. We further develop our model to generalize the resonant time-scale operation, and provide responses for square and triangular voltage waveforms as two examples. To this end, we develop a general tool that uses Fourier analysis to construct CDI effluent dynamics for arbitrary input waveforms. Using this tool, we show that most of the salt removal (∼95%) for square and triangular voltage forcing waveforms is achieved by the fundamental Fourier (sinusoidal) mode. The frequency of higher Fourier modes precludes high flow efficiency for these modes, so these modes consume additional energy for minimal additional salt removed. This deficiency of higher frequency modes further highlights the advantage of DC-offset sinusoidal forcing for CDI operation.

The in-plane permeability of porous thin films is an important fluid mechanical property that determines wicking and pressure-driven flow behavior in such materials. This property has so far been challenging to measure directly due to the small sidewall cross-sectional area of thin films available for flow. In this work, we propose and experimentally demonstrate a novel technique for directly measuring in-plane permeability of porous thin films of arbitrary thicknesses, in situ, using a manifold pressed to the top surface of the film. We both measure and simulate the influence of the two dimensional flow field produced in a film by the manifold and extract the permeability from measurements of pressure drop at fixed flow rates. Permeability values measured using the technique for a periodic array of channels are comparable to theoretical predictions. We also determine in-plane permeability of arrays of pillars and electrodeposited porous copper films. This technique is a robust tool to characterize permeability of thin films of arbitrary thicknesses on a variety of substrates. In Supplementary material, we provide a solid model, which is useful in three-dimensional printer reproductions of our device.

We present a simple, top-down approach for the calculation of minimum energy consumption of electrosorptive ion separation using variational form of the (Gibbs) free energy. We focus and expand on the case of electrostatic capacitive deionization (CDI), and the theoretical framework is independent of details of the double-layer charge distribution and is applicable to any thermodynamically consistent model, such as the Gouy-Chapman-Stern (GCS) and modified Donnan (mD) models. We demonstrate that, under certain assumptions, the minimum required electric work energy is indeed equivalent to the free energy of separation. Using the theory, we define the thermodynamic efficiency of CDI. We explore the thermodynamic efficiency of current experimental CDI systems and show that these are currently very low, less than 1% for most existing systems. We applied this knowledge and constructed and operated a CDI cell to show that judicious selection of the materials, geometry, and process parameters can be used to achieve a 9% thermodynamic efficiency (4.6 kT energy per removed ion). This relatively high value is, to our knowledge, by far the highest thermodynamic efficiency ever demonstrated for CDI. We hypothesize that efficiency can be further improved by further reduction of CDI cell series resistances and optimization of operational parameters.

Ion transport in porous conductive materials is of great importance in a variety of electrochemical systems including batteries and supercapacitors. We here analyze the coupling of flow and charge transport and charge capacitance in capacitive deionization (CDI). In CDI, a pair of porous carbon electrodes is employed to electrostatically retain and remove ionic species from aqueous solutions. We here develop and solve a novel unsteady two-dimensional model for capturing the ion adsorption/desorption dynamics in a flow-between CDI system. We use this model to study the complex, nonlinear coupling between electromigration, diffusion, and advection of ions. We also fabricated a laboratory-scale CDI cell which we use to measure the near-equilibrium, cumulative adsorbed salt, and electric charge as a function of applied external voltage. We use these integral measures to validate and calibrate this model. We further present a detailed computational study of the spatiotemporal adsorption/desorption dynamics und...

Capacitive deionization (CDI) is a promising desalination technology, which operates at low pressure, low temperature, requires little infrastructure, and has the potential to consume less energy for brackish water desalination. However, CDI devices consume significantly more energy than the theoretical thermodynamic minimum, and this is at least partly due to resistive power dissipation. We here report our efforts to characterize electric resistances in a CDI system, with a focus on the resistance associated with the contact between current collectors and porous electrodes. We present an equivalent circuit model to describe resistive components in a CDI cell. We propose measurable figures of merit to characterize cell resistance. We also show that contact pressure between porous electrodes and current collectors can significantly reduce contact resistance. Lastly, we propose and test an alternative electrical contact configuration which uses a pore-filling conductive adhesive (silver epoxy) and achieves significant reductions in contact resistance.

Telomere extension has been proposed as a means to improve cell culture and tissue engineering and to treat disease. However, telomere extension by nonviral, nonintegrating methods remains inefficient. Here we report that delivery of modified mRNA encoding TERT to human fibroblasts and myoblasts increases telomerase activity transiently (24–48 h) and rapidly extends telomeres, after which telomeres resume shortening. Three successive transfections over a 4 d period extended telomeres up to 0.9 kb in a cell type-specific manner in fibroblasts and myoblasts and conferred an additional 28 ± 1.5 and 3.4 ± 0.4 population doublings (PDs), respectively. Proliferative capacity increased in a dose-dependent manner. The second and third transfections had less effect on proliferative capacity than the first, revealing a refractory period. However, the refractory period was transient as a later fourth transfection increased fibroblast proliferative capacity by an additional 15.2 ± 1.1 PDs, similar to the first transfection. Overall, these treatments led to an increase in absolute cell number of more than 1012-fold. Notably, unlike immortalized cells, all treated cell populations eventually stopped increasing in number and expressed senescence markers to the same extent as untreated cells. This rapid method of extending telomeres and increasing cell proliferative capacity without risk of insertional mutagenesis should have broad utility in disease modeling, drug screening, and regenerative medicine.—Ramunas, J., Yakubov, E., Brady, J. J., Corbel, S. Y., Holbrook, C., Brandt, M., Stein, J., Santiago, J. G., Cooke, J. P., Blau, H. M. Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells.

BackgroundEukaryotes transcribe RNAs in nuclei and transport them to the cytoplasm through multiple steps of post-transcriptional regulation. Existing single-cell sequencing technologies, however, are unable to analyse nuclear (nuc) and cytoplasmic (cyt) RNAs separately and simultaneously. Hence, there remain challenges to discern correlation, localisation, and translocation between them.ResultsHere we report a microfluidic system that physically separates nucRNA and cytRNA from a single cell and enables single-cell integrated nucRNA and cytRNA-sequencing (SINC-seq). SINC-seq constructs two individual RNA-seq libraries, nucRNA and cytRNA per cell, quantifies gene expression in the subcellular compartments and combines them to create a novel single-cell RNA-seq data enabled by our system, which we here term in-silico single cell.ConclusionsLeveraging SINC-seq, we discovered three distinct natures of correlation among cytRNA and nucRNA that reflected the physiological state of single cells: The cell-cycle-related genes displayed highly correlated expression pattern with minor differences; RNA splicing genes showed lower nucRNA-to-cytRNA correlation, suggesting a retained intron may be implicated in inhibited mRNA transport; A chemical perturbation, sodium butyrate treatment, transiently distorted the correlation along differentiating human leukemic cells to erythroid cells. These data uniquely provide insights into the regulatory network of mRNA from nucleus toward cytoplasm at the single cell level.

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.

In this paper, we are interested in the design of a microfluidic mixer based on hydrodynamic focusing which is used to initiate the folding process (i.e., changes of the molecular structure) of a protein by diluting a protein solution to decrease its denaturant concentration to a given value in a short time interval we refer to as mixing time. Our objective is to optimize this mixer by choosing suitable shape and flow conditions in order to minimize its mixing time. To this end, we first introduce a numerical model that enables computation of the mixing time of a considered mixer. Then, we define a mixer optimization problem and solve it using a hybrid global optimization algorithm.

We present an experimental study of coupling of isotachophoresis (ITP) and affinity chromatography (AC) to effect rapid, selective purification with high column utilization and high resolution. We provide a detailed protocol for performing ITP-AC and describe the design of a buffer system to perform sequence specific separation of nucleic acids. We describe the synthesis and functionalization of our affinity substrate, poly(glycidyl methacrylate-co-ethylene dimethacrylate) porous polymer monolith (GMA-EDMA PPM). This substrate allows easy immobilization of affinity probes, is nonsieving (even to macromolecules), and exhibits negligible nonspecific binding. We demonstrate ITP-AC with 25 nt, Cy5 labeled DNA target and a DNA probe and study the spatiotemporal dynamics using epifluorescence imaging. We make qualitative and quantitative comparisons between these data and the model presented in the first part of this two-paper series. We vary the target concentration from 1 pg μL(-1) to 100 pg μL(-1) and ITP velocity over the range of 10-50 μm s(-1), and thereby explore over 4 orders of magnitude of scaled target amount. We observe very good agreement between predictions and experimental data for the spatiotemporal behavior of the coupled ITP and affinity process, and for key figures of merit, including scaled capture length and maximum capture efficiency. Lastly, we demonstrate that the resolution of ITP-AC increases linearly with time and purify 25 nt target DNA from 10,000-fold higher abundance background (contaminating) genomic fish sperm DNA. We perform this capture from 200 μL of sample in under 1 mm column length and within10 min.

Reviewed are methods of nucleic acid (NA) extraction and sample preparation using an electrophoretic purification and focusing method called isotachophoresis (ITP). ITP requires no special surface chemistries or geometric structures, and can be achieved in a compact system with no moving parts. ITP is also compatible with a wide range of samples and lysing methods. Described are general principles of ITP, considerations around the application of ITP to biological samples (e.g., blood, urine and saliva), ITP electrolyte design considerations for fast and selective NA purification, and examples of ITP compatible lysing methods. Several of the challenges associated with purification of NAs are presented as well as methods to address these. Lastly, specific examples of lysing methods and ITP chemistries are described for purification of NA including host and pathogenic DNA, pathogenic rRNA, and host micro-RNA from complex sample matrices.

We present a novel microchip device for purification of nucleic acids from 25μL biological samples using isotachophoresis (ITP). The device design incorporates a custom capillary barrier structure to facilitate robust sample loading. The chip uses a 2mm channel width and 0.15mm depth to reduce processing time, mitigate Joule heating, and achieve high extraction efficiency. To reduce pH changes in the device due to electrolysis, we incorporated a buffering reservoir physically separated from the sample output reservoir. To reduce dispersion of the ITP-focused zone, we used optimized turn geometries. The chip was fabricated by injection molding PMMA and COC plastics through a commercial microfluidic foundry. The extraction efficiency of nucleic acids from the device was measured using fluorescent quantification, and an average recovery efficiency of 81% was achieved for nucleic acid masses between 250pg and 250ng. The devices were also used to purify DNA from whole blood, and the extracted DNA was amplified using qPCR to show the PCR compatibility of the purified sample.

We present an on-chip electrokinetic method to increase the reaction kinetics and sensitivity of DNA microarray hybridization. We use isotachophoresis (ITP) to preconcentrate target molecules in solution and transport them over the immobilized probe sites of a microarray, greatly increasing the binding reaction rate. We show theoretically and experimentally that ITP-enhanced microarrays can be hybridized much faster and with higher sensitivity than conventional methods. We demonstrate our assay using a microfluidic system consisting of a PDMS microchannel superstructure bonded onto a glass slide on which 60 spots of 20-27 nt ssDNA oligonucleotide probes are immobilized. Our 30 min assay results in an 8.2 fold higher signal than the conventional overnight hybridization at 100 fM target concentration. We show rapid and quantitative detection over 4 orders of magnitude dynamic range of target concentration with no increase in the nonspecific signal. Our technique can be further multiplexed for higher density microarrays and extended for other reactions of target-surface immobilized ligands.

Capacitive deionization (CDI) is a promising technique for salt removal and may have potential for highly selective removal of ion species. In this work, we take advantage of functional groups usually used with ionic exchange resins and apply these to CDI. To this end, we functionalize activated carbon with a quaternary amines surfactant and use this surface to selectively and passively (no applied field) trap nitrate ions. We then set the cell voltage to a constant value to regenerate these electrodes, resulting in an inverted capacitive deionization (i-CDI) operation. Unlike resins, we avoid use of concentrated chemicals for regeneration. We measure the selectivity of nitrate versus chloride ions as a function of regeneration voltage and initial chloride concentration. We experimentally demonstrate up to about 6.5-fold (observable) selectivity in a cycle with a regeneration voltage of 0.4 V. We also demonstrate a novel multi-pass, air-flush i-CDI operation to selectively enrich nitrate with high water recovery. We further present a dynamic, multi-species electrosorption and equilibrium solution-to-surface chemical reaction model and validate the model with detailed measurements. Our i-CDI system exhibits higher nitrate selectivity at lower voltages; making it possible to reduce NaNO3 concentrations from ∼170 ppm to below the limit of maximum allowed values for nitrate in drinking water of about 50 ppm NaNO3., Graphical abstract Image 1, Highlights • Model and experiment validation for dynamic multi-ion electrosorption and selectivity. • Inverted capacitive deionization for 6.5-fold nitrate selectivity, 0.4 V regeneration. • Demonstrated multi-pass gas-flush system for ion enrichment and high water recovery.

We present a simple-to-implement method for analyzing images of randomly distributed particles transported through a fluidic channel. We term this method particle imaging, tracking and collocation (PITC). Our method uses off-the-shelf optics including a CCD camera, epifluorescence microscope, and a dual-view color separator to image freely suspended particles in a wide variety of microchannels (with optical access for image collection). The particles can be transported via electrophoresis and/or pressure driven flow to increase throughput of analysis. We here describe the implementation of the algorithm and demonstrate and validate three of its capabilities: (1) identification of particle coordinates, (2) tracking of particle motion, and (3) monitoring of particle interaction via collocation analysis. We use Monte Carlo simulations for validation and optimization of the input parameters. We also present an experimental demonstration of the analysis on challenging image data, including a flow of two, interacting Brownian particle populations. In the latter example, we use PITC to detect the presence of target DNA by monitoring the hybridization-induced binding between the two populations of beads, each functionalized with DNA probes complementary to the target molecule.

Porous electrode capacitors are used extensively in systems which store energy, harvest mixing energy, or desalinate water. These electrodes can possess a hierarchical pore structure with larger macroscale pores allowing for facile ion and fluid transport, and smaller, nanometer-scale pores enabling significant ion storage. We here present a combined theoretical (linear circuit model) and experimental (electrochemical impedance spectroscopy) study of porous carbon electrode capacitors which integrate nanoscale pores into a micron-scale porous network. Our experiments are performed on a set of customfabricated hierarchical carbon aerogel electrodes with varying pore structure, including electrodes with sub-nanometer (sub-nm) pores. Our combined theory and experimental approach allows us to demonstrate the utility of our model, perform detailed characterizations of our electrodes, study the effects of pore structure variations on impedance, and propose hierarchical electrode design and characterization guidelines. Further, we demonstrate that our approach is promising toward the detailed study of ion storage mechanisms in sub-nm pores.

Focusing in on the small: A two-stage microRNA detection assay uses electrokinetic focusing to speed up hybridization and a functionalized hydrogel for affinity purification. This method detects microRNAs in 15 minutes with single-nucleotide specificity, and processes only 5 ng of total RNA.

We explored the energy loss mechanisms in capacitive deionization (CDI). We hypothesize that resistive and parasitic losses are two main sources of energy losses. We measured contribution from each loss mechanism in water desalination with constant current (CC) charge/discharge cycling. Resistive energy loss is expected to dominate in high current charging cases, as it increases approximately linearly with current for fixed charge transfer (resistive power loss scales as square of current and charging time scales as inverse of current). On the other hand, parasitic loss is dominant in low current cases, as the electrodes spend more time at higher voltages. We built a CDI cell with five electrode pairs and standard flow between architecture. We performed a series of experiments with various cycling currents and cut-off voltages (voltage at which current is reversed) and studied these energy losses. To this end, we measured series resistance of the cell (contact resistances, resistance of wires, and resistance of solution in spacers) during charging and discharging from voltage response of a small amplitude AC current signal added to the underlying cycling current. We performed a separate set of experiments to quantify parasitic (or leakage) current of the cell versus cell voltage. We then used these data to estimate parasitic losses under the assumption that leakage current is primarily voltage (and not current) dependent. Our results confirmed that resistive and parasitic losses respectively dominate in the limit of high and low currents. We also measured salt adsorption and report energy-normalized adsorbed salt (ENAS, energy loss per ion removed) and average salt adsorption rate (ASAR). We show a clear tradeoff between ASAR and ENAS and show that balancing these losses leads to optimal energy efficiency.

We here present an approach to cooling of electronics requiring dissipation of extreme heat fluxes exceeding 1 kW/cm2 over ∼1 cm2 areas. The approach applies a combination of heat spreading using laser micromachined diamond heat sinks; evaporation/boiling in fine featured (5 µm) conformal porous copper coatings; microfluidic liquid routing for uniform coolant supply over the surface of the heat sink; and phase separation to control distribution of liquid and vapor phases. We characterize the performance of these technologies independently and integrated into functional devices. We report two-phase heat transfer performance of diamond/porous copper heat sinks with microfluidic manifolding at full device scales (0.7 cm2) with heat fluxes exceeding 1300 W/cm2 using water working fluid. We further show application of hydrophobic phase separation membranes for phase management with heat dissipation exceeding 450 W/cm2 at the scale of a single extended surface (∼300 µm).

We consider a particular hydrodynamic focusing microfluidic mixer used to initiate the folding process of individual proteins, which has been designed in a previous work and exhibited a mixing time of 0.1 μs. The aim of the current paper is twofold. First, we explore the sensitivity of mixing time to key geometric and flow parameters. In particular, we study the angle between inlets, the shape of the channel intersections, channel widths, mixer depth, mixer symmetry, inlet velocities, working fluid physical properties, and denaturant concentration thresholds. Second, we analyze the uniformity of mixing times as a function of inlet flow streamlines. We find the shape of the intersection, channel width, inlet velocity ratio, and asymmetries have strong effects on mixing time; while inlet angles, mixer depth, fluid properties, and concentration thresholds have weaker effects. Also, the uniformity of the mixing time is preserved for most of the inlet flow and distances of down to within about 0.4 μm of the mixer wall. We offer these analyses of sensitivities to imperfections in mixer geometry and flow conditions as a guide to experimental efforts which aim to fabricate and use these types of mixers. Our study also highlights key issues and provides a guide to the optimization and practical design of other microfluidic devices dependent on both geometry and flow conditions.

We present an experimental study on the effect of polymer PVP on EOF mobility of microchannels wet etched into optical white soda lime glass, also known as Crown glass. We performed experiments to evaluate the effect of PVP concentration and pH on EOF mobility. We used on-chip capillary zone electrophoresis and a neutral fluorescent dye as a passive marker to quantify the electroosmotic flow. We performed experiments under controlled conditions by varying pH from 5.2 and 10.3 and concentration of PVP from 0 to 2.0% w/w at constant ionic strength (30 mM). Our experiments show that PVP at concentrations of 1.0% or above very effectively suppress EOF at low pH (6.6). At high pH of 10.3, PVP has a much weaker suppressing effect on EOF and increasing its concentration above about 0.5% showed negligible effect on EOF mobility. Finally, we briefly discuss the effects of pH on using PVP as an adsorbed coating. Our experiments provide useful guidelines on choosing correct pH and concentration of PVP for effective EOF suppression in glass channels.

Preparation of raw, untreated biological samples remains a major challenge in microfluidics. We present a novel microfluidic device based on the integration of printed circuit boards and an isotachophoresis assay for sample preparation of nucleic acids from biological samples. The device has integrated resistive heaters and temperature sensors as well as a 70 μm × 300 μm × 3.7 cm microfluidic channel connecting two 15 μL reservoirs. We demonstrated this device by extracting pathogenic nucleic acids from 1 μL dispensed volume of whole blood spiked with Plasmodium falciparum. We dispensed whole blood directly onto an on-chip reservoir, and the system's integrated heaters simultaneously lysed and mixed the sample. We used isotachophoresis to extract the nucleic acids into a secondary buffer via isotachophoresis. We analyzed the convective mixing action with micro particle image velocimetry (micro-PIV) and verified the purity and amount of extracted nucleic acids using off-chip quantitative polymerase chain reaction (PCR). We achieved a clinically relevant limit of detection of 500 parasites per microliter. The system has no moving parts, and the process is potentially compatible with a wide range of on-chip hybridization or amplification assays.

We present a model and an associated numerical scheme to simulate complex electrokinetic processes in channels with nonuniform cross-sectional area. We develop a quasi-1D model based on local cross-sectional area averaging of the equations describing unsteady, multispecies, electromigration-diffusion transport. Our approach uses techniques of lubrication theory to approximate electrokinetic flows in channels with arbitrary variations in cross-section; and we include chemical equilibrium calculations for weak electrolytes, Taylor-Aris type dispersion due of nonuniform bulk flow, and the effects of ionic strength on species mobility and on acid-base equilibrium constants. To solve the quasi-1D governing equations, we provide a dissipative finite volume scheme that adds numerical dissipation at selective locations to ensure both unconditional stability and high accuracy. We couple the numerical scheme with a novel adaptive grid refinement algorithm that further improves the accuracy of simulations by minimizing numerical dissipation. We benchmark our numerical scheme with existing numerical schemes by simulating nonlinear electrokinetic problems, including ITP and electromigration dispersion in CZE. Simulation results show that our approach yields fast, stable, and high-resolution solutions using an order of magnitude less grid points compared to the existing dissipative schemes. To highlight our model's capabilities, we demonstrate simulations that predict increase in detection sensitivity of ITP in converging cross-sectional area channels. We also show that our simulations of ITP in variable cross-sectional area channels have very good quantitative agreement with published experimental data.

We introduce an on-chip electrokinetic assay to perform high-sensitivity nucleic acid (NA) detection. This assay integrates electrokinetic sample focusing using isotachophoresis (ITP) with a background signal-removal strategy that employs photopatterened, DNA-functionalized hydrogels. In this multistage assay, ITP first enhances hybridization kinetics between target NAs and end-labeled complementary reporters. After enhanced hybridization, migration through a DNA-functionalized hydrogel region removes excess reporters through affinity interactions. We demonstrate our assay on microRNAs, an important class of low-abundance biomarkers. The assay exhibits 4 orders of magnitude dynamic range, near 1 pM detection limits starting from less than 100 fg of microRNA, and high selectivity for mature microRNA sequences, all within a 10 min run time. This new microfluidic framework provides a unique quantitative assay for NA detection.

We demonstrate a novel assay for physicochemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected with Pseudomonas putida . This on-chip assay is unique in that the extraction can be automated using isotachophoresis in a simple device with no moving parts, it protects RNA from degradation when isolating from ribonuclease-rich matrices (such as blood), and produces a purified total nucleic acid sample that is compatible with enzymatic amplification assays. We show that the purified RNA is compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR.

We use isotachophoresis (ITP) to control and increase the rate of nucleic acid hybridization reactions in free solution. We present a new physical model, validation experiments, and demonstrations of this assay. We studied the coupled physicochemical processes of preconcentration, mixing, and chemical reaction kinetics under ITP. Our experimentally validated model enables a closed form solution for ITP-aided reaction kinetics, and reveals a new characteristic time scale which correctly predicts order 10,000-fold speed-up of chemical reaction rate for order 100 pM reactants, and greater enhancement at lower concentrations. At 500 pM concentration, we measured a reaction time which is 14,000-fold lower than that predicted for standard second-order hybridization. The model and method are generally applicable to acceleration of reactions involving nucleic acids, and may be applicable to a wide range of reactions involving ionic reactants.

We present a novel method of creating concentration cascade of leading electrolyte (LE) in isotachophoresis (ITP) by using bidirectional ITP. ITP establishes ion-concentration shock waves between high-mobility LE and low-mobility trailing electrolyte (TE) ions. In bidirectional ITP, we set up simultaneous shock waves between anions and cations such that these waves approach each other and interact. The shock interaction causes a sudden decrease in LE concentration ahead of the focused anions and a corresponding decrease in analyte zone concentrations. This readjustment of analyte zone concentrations is accompanied by a corresponding increase in their zone lengths, in accordance to conservation laws. The method generates in situ gradient in the LE concentration, and therefore can be achieved in a single, straight channel simply by establishing the initial electrolyte chemistry. We have developed an analytical model useful in designing the process for maximum sensitivity and estimating increase in sample zone length due to shock interaction. We also illustrate the technique and evaluate its effectiveness in increasing detection sensitivity using transient simulations of species transport equations. We validated the theoretical predictions using experimental visualizations of bidirectional ITP zones for various electrolyte chemistries. Lastly, we use our technique to demonstrate a factor of 20 increase in the sensitivity of ITP-based detection of 2,4,6-trichlorophenol.

We demonstrate a technique for purification of nucleic acids from malaria parasites infecting human erythrocytes using isotachophoresis (ITP). We release nucleic acids from malaria-infected erythrocytes by lysing with heat and proteinase K for 10 min and immediately, thereafter, load sample onto a capillary device. We study the effect of temperature on lysis efficiency. We also implement pressure-driven counterflow during ITP extraction to extend focusing time and increase nucleic acid yield. We show that the purified genomic DNA samples are compatible with polymerase chain reaction (PCR) and demonstrate a clinically relevant limit of detection of 0.5 parasites per nanoliter using quantitative PCR.

We present an experimental study of the effect of pH, ionic strength, and concentrations of the electroosmotic flow (EOF)-suppressing polymer polyvinylpyrrolidone (PVP) on the electrophoretic mobilities of commonly used fluorescent dyes (fluorescein, Rhodamine 6G, and Alexa Fluor 488). We performed on-chip capillary zone electrophoresis experiments to directly quantify the effective electrophoretic mobility. We use Rhodamine B as a fluorescent neutral marker (to quantify EOF) and CCD detection. We also report relevant acid dissociation constants and analyte diffusivities based on our absolute estimate (as per Nernst-Einstein diffusion). We perform well-controlled experiments in a pH range of 3-11 and ionic strengths ranging from 30 to 90 mM. We account for the influence of ionic strength on the electrophoretic transport of sample analytes through the Onsager and Fuoss theory extended for finite radii ions to obtain the absolute mobility of the fluorophores. Lastly, we briefly explore the effect of PVP on adsorption-desorption dynamics of all three analytes, with particular attention to cationic R6G.

We present a novel technique for coupling isotachophoretic preconcentration and electrophoretic separation using bidirectional isotachophoresis (ITP). Bidirectional ITP simultaneously sets up sharp ITP interfaces between relatively high- and low-mobility cations and high- and low-mobility anions. These two interfaces can migrate toward each other and be described as ion concentration shock waves. We here demonstrate a bidirectional ITP process in which we use the interaction of these anionic and cationic ITP shock waves to trigger a transformation from ITP preconcentration to electrophoretic separation. We use anionic ITP to focus anionic sample species prior to shock interaction. The interaction of the counter-propagating anionic and cationic ITP shocks then changes the local pH (and ionic strength) of the focused analyte zones. Under this new condition, the analytes no longer focus and begin to separate electrophoretically. The method provides faster and much less dispersive transition from ITP preconcentration to electrophoretic separation compared with traditional (unidirectional) transient ITP. It eliminates the need for intermediate steps between focusing and separation, such as manual buffer exchanges. We illustrate the technique with numerical simulations of species transport equations. We have validated our simulations with experimental visualization of bidirectional ITP zones. We then show the effectiveness of the technique by coupling ITP preconcentration and high-resolution separation of a 1 kbp DNA ladder via shock interaction in bidirectional ITP.

We present an analytical, numerical and experimental study of advective dispersion in isotachophoresis (ITP). We analyse the dynamics of the concentration field of a focused analyte in peak mode ITP. The analyte distribution is subject to electromigration, diffusion and advective dispersion. Advective dispersion results from strong internal pressure gradients caused by non-uniform electro-osmotic flow (EOF). Analyte dispersion strongly affects the sensitivity and resolution of ITP-based assays. We perform axisymmetric time-dependent numerical simulations of fluid flow, diffusion and electromigration. We find that analyte properties contribute greatly to dispersion in ITP. Analytes with mobility values near those of the trailing (TE) or leading electrolyte (LE) show greater penetration into the TE or LE, respectively. Local pressure gradients in the TE and LE then locally disperse these zones of analyte penetration. Based on these observations, we develop a one-dimensional analytical model of the focused sample zone. We treat the LE, TE and LE–TE interface regions separately and, in each, assume a local Taylor–Aris-type effective dispersion coefficient. We also performed well-controlled experiments in circular capillaries, which we use to validate our simulations and analytical model. Our model allows for fast and accurate prediction of the area-averaged sample distribution based on known parameters including species mobilities, EO mobility, applied current density and channel dimensions. This model elucidates the fundamental mechanisms underlying analyte advective dispersion in ITP and can be used to optimize detector placement in detection-based assays.

We present a novel assay for rapid detection and identification of bacterial urinary tract infections using isotachophoresis (ITP) and molecular beacons. We applied on-chip ITP to extract and focus 16S rRNA directly from bacterial lysate and used molecular beacons to achieve detection of bacteria specific sequences. We demonstrated detection of E. coli in bacteria cultures as well as in patient urine samples in the clinically relevant range 1E6-1E8 cfu/mL. For bacterial cultures we further demonstrate quantification in this range. The assay requires minimal sample preparation (a single centrifugation and dilution), and can be completed, from beginning of lysing to detection, in under 15 min. We believe that the principles presented here can be used for design of other rapid diagnostics or detection methods for pathogenic diseases.

We here present and demonstrate a novel technique based on isotachophoresis (ITP) for the quantification of global microRNA (miRNA) abundance in total RNA. We leverage the selectivity of ITP to concentrate miRNA and exclude longer RNA molecules from the focused zone. We designed a novel ITP strategy where we initially establish three contiguous zones of sieving polymer, electrolyte, and denaturant concentrations. This allows for successive preconcentration, selection, and detection of miRNA. We optimized chemistry in each zone for high sensitivity and exquisite selectivity for miRNA. This technique allows for the measurement of the total miRNA content in a sample and its comparison between different cell types and tissues. We demonstrated and validated the efficacy of this technique by comparing global miRNA abundance in subconfluent and confluent cell cultures.

We report the development and experimental analysis of a two-liquid electroosmotic (EO) pump. The pump uses a collapsible and impermeable membrane to separate the liquid delivered from a working electrolyte solution optimized for EO pumping. To achieve high pressures at low voltages, we use a porous glass substrate with 450 nm mean diameter pores and a 1 mM sodium borate electrolyte. Results show that the membranes add negligible pressure load and that the pump can deliver adequately stable flow rates after an initial transient period. At flow rates and pressures suitable for drug delivery (order 10 μl/min and a few kPa), the pump requires an applied voltage and power of less than 3 V and 75 μW, respectively. Based on the relative scaling of flow rate, current, and time, we hypothesize that distinct initial transients in flow rate are attributable to concentration polarization and its effects on the spatiotemporal ionic strength of the working fluid.

In a typical air-breathing fuel cell design, ambient air is supplied to the cathode by natural convection and dry hydrogen is supplied to a dead-ended anode. While this design is simple and attractive for portable low-power applications, the difficulty in implementing effective and robust water management presents disadvantages. In particular, excessive flooding of the open-cathode during long-term operation can lead to a dramatic reduction of fuel cell power. To overcome this limitation, we report here on a novel airbreathing fuel cell water management design based on a hydrophilic and electrically conductive wick in conjunction with an electroosmotic (EO) pump that actively pumps water out of the wick. Transient experiments demonstrate the ability of the EO-pump to “resuscitate” the fuel cell from catastrophic flooding events, while longer term galvanostatic measurements suggest that the design can completely eliminate cathode flooding using less than 2% of fuel cell power, and lead to stable operation with higher net power performance than a control design without EO-pump. This demonstrates that active EO-pump water management, which has previously only been demonstrated in forced-convection fuel cell systems, can also be applied effectively to miniaturized (

Water management is a significant challenge in portable polymer electrolyte membrane (PEM) fuel cells and particularly in proton exchange membrane (PEM) fuel cells with air-breathing cathodes. Liquid water condensation and accumulation at the cathode surface is unavoidable in a passive design operated over a wide range of ambient and load conditions. Excessive flooding or dry out of the open cathode can lead to a dramatic reduction of fuel cell power. We report a water management design based on a hydrophilic and electrically conductive wick. A prototype air-breathing fuel cell with the proposed water management design successfully operated under severe flooding conditions, ambient temperature 10 ◦ C and relative humidity of 80%, for up to 6 h with no observable cathode flooding or loss of performance.

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.

We present a numerical and experimental study of the effects of ionic strength on electrophoretic focusing and separations. We review the development of ionic strength models for electrophoretic mobility and chemical activity and highlight their differences in the context of electrophoretic separation and focusing simulations. We couple a fast numerical solver for electrophoretic transport with the Onsager-Fuoss model for actual ionic mobility and the extended Debye-Huckle theory for correction of ionic activity. Model predictions for fluorescein mobility as a function of ionic strength and pH compare well with data from CZE experiments. Simulation predictions of preconcentration factors in peak mode ITP also compare well with the published experimental data. We performed ITP experiments to study the effect of ionic strength on the simultaneous focusing and separation. Our comparisons of the latter data with simulation results at 10 and 250 mM ionic strength show the model is able to capture the observed qualitative differences in ITP analyte zone shape and order. Finally, we present simulations of CZE experiments where changes in the ionic strength result in significant change in selectivity and order of analyte peaks. Our simulations of ionic strength effects in capillary electrophoresis compare well with the published experimental data.

Air-delivery is typically the largest parasitic loss in PEM fuel cell systems. We develop a passive water management system that minimizes this loss by enabling stable, flood-free performance in parallel channel architectures, at very low air stoichiometries. Our system employs in situ -polymerized wicks which conform to and coat cathode flow field channel walls, thereby spatially defining regions for water and air transport. We first present the fabrication procedure, which incorporates a flow field plate geometry comparable to many state-of-the-art architectures (e.g., stamped metal or injection molded flow fields). We then experimentally compare water management flow field performance versus a control case with no wick integration. At the very low air stoichiometry of 1.15, our system delivers a peak power density of 0.68 W cm −2 . This represents a 62% increase in peak power over the control case. The open channel and manifold geometries are identical for both cases, and we demonstrate near identical inlet-to-outlet cathode pressure drops at all fuel cell operating points. Our water management system therefore achieves significant performance enhancement without introducing additional parasitic losses.

We present a novel method for identification of unlabeled analytes using fluorescent carrier ampholytes and isotachophoresis (ITP). The method is based on previous work where we showed that the ITP displacement of carrier ampholytes can be used for detection of unlabeled (nonfluorescent) analytes. We here propose a signal analysis method based on integration of the associated fluorescent signal. We define a normalized signal integral which is equivalent to an accurate measure of the amount of carrier ampholytes which are focused between the leading electrolyte and the analyte. We show that this parameter can be related directly to analyte effective mobility. Using several well characterized analytes, we construct calibration curves relating effective mobility and carrier ampholyte displacement at two different leading electrolyte (LE) buffers. On the basis of these calibration curves, we demonstrate the extraction of fully ionized mobility and dissociation constant of 2-nitrophenol and 2,4,6-trichlorophenol from ITP experiments with fluorescent carrier ampholytes. This extraction is based on no a priori assumptions or knowledge of these two toxic chemicals. This technique allows simultaneous identification of multiple analytes by their physiochemical properties in a few minutes and with no sample preparation.

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.

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. In the CP regime, EO pump models likely fail as these models do not account for the strong spatiotemporal variations in ionic strength. 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.

We present theoretical and experimental studies of an active water management system for proton exchange membrane (PEM) fuel cells that uses integrated wicks and electroosmotic (EO) pumps. The wicks and EO pumps act in concert to remove problematic excess liquid water from the fuel cell. In a previous paper, we showed that this system increases maximum power density by as much as 60% when operating with low air stoichiometric ratios and a parallel channel flow field. The theoretical model we develop here accounts for several key factors specific to optimizing system performance, including the wick’s hydraulic resistance, the variation of water pH, and the EO pump’s electrochemical reactions. We use this model to illustrate the favorable scaling of EO pumps with fuel cells for water management. In the experimental portion of this study, we prevent flooding by applying a constant voltage to the EO pump. We experimentally analyze the relationships between applied voltage, pump performance, and fuel cell performance. Further, we identify the minimum applied pump voltage necessary to prevent flooding. This study has wide applicability as it also identifies the relationship between active water removal rate and flooding prevention. © 2009 Elsevier Ltd. All rights reserved.

We present and demonstrate a novel technique for the purification of nucleic acids from biological samples using isotachophoresis (ITP). We demonstrate a simple and rapid method to achieve ITP-based extraction, preconcentration, and purification of DNA from nanoliter volumes of whole blood. We show that ITP purification yields genomic DNA samples which can be quantitated with fluorescence measurements and are immediately compatible with polymerase chain reaction (PCR) (e.g., a PCR-friendly solution free of significant inhibitors). We hypothesize ITP purification is applicable to processing of a wide range of complex biological samples.

We present controlled experiments on a miniature direct methanol fuel cell (DMFC) to study the effects of methanol flow rate, current density, and void fraction on pressure drop across the DMFC anode. We also present an experimental technique to measure void fraction, liquid slug length, and velocity of the two-phase slug flow exiting the DMFC. For our channel geometry in which the diameter of the largest inscribed sphere (a) is 500 μm, pressure drop scales with the number of gas slugs in the channel, surface tension, and a. This scaling demonstrates the importance of capillary forces in determining the hydrodynamic characteristics of the DMFC anode. This work is aimed at aiding the design of fuel pumps and anode flow channels for miniature DMFC systems.