Web-Based Open-Source Tool for Isotachophoresis.

We present the development of a client-side web-based simulator for complex electrophoresis phenomena, including isotachophoresis. The simulation tool is called Client-based Application for Fast Electrophoresis Simulation (CAFES). CAFES uses the broad cross-browser compatibility of JavaScript to provide a rapid and easy-to-use tool for coupled unsteady electromigration, diffusion, and equilibrium electrolyte reactions among multiple weak electrolytes. The code uses a stationary grid (for simplicity) and an adaptive time step to provide reliable estimates of ion concentration dynamics (including pH profile evolution), requiring no prior installation nor compilation. CAFES also offers a large database of commonly used species and their relevant physicochemical properties. We present a validation of predictions from CAFES by comparing them to experimental data of peak- and plateau-mode isotachophoresis experiments. The code yields accurate estimates of interface velocity, plateau length and relative intensity, and pH variations while significantly reducing the computation time compared to existing codes. The tool is open-source and available for free at https://microfluidics.stanford.edu/cafes.

Coupling isotachophoresis and capillary electrophoresis: a review and comparison of methods.

We present a comprehensive review and comparison of the methodologies for increasing sensitivity and resolution of capillary electrophoresis (CE) using online transient isotachophoresis (tITP). We categorize the diverse set of coupled tITP and CE (tITP-CE) methods based on their fundamental principles for disrupting isotachophoretic preconcentration and triggering electrophoretic separation. Based on this classification, we discuss important features, advantages, limitations, and optimization principles of various tITP-CE methods. We substantiate our discussion with original simulations, instructive examples, and published experimental results.

Integration of rapid DNA hybridization and capillary zone electrophoresis using bidirectional isotachophoresis.

We present a method for rapid, sequence-specific detection of multiple DNA fragments by integrating isotachophoresis (ITP) based DNA hybridization and capillary zone electrophoresis (CZE) using bidirectional ITP. Our method leverages the high preconcentration ability of ITP to accelerate slow, second-order DNA hybridization kinetics, and the high resolving power of CZE to separate and identify reaction products. We demonstrate the speed and sensitivity of our assay by detecting 5 pM, 39 nt ssDNA target within 3 min, using a molecular beacon probe. We also demonstrate the feasibility of our assay for multiplexed detection of multiple-length ssDNA targets by simultaneously detecting 39 and 90 nt ssDNA targets.

Effect of PVP on the electroosmotic mobility of wet-etched glass microchannels.

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.

Robust and high-resolution simulations of nonlinear electrokinetic processes in variable cross-section channels.

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.

Desalination and hydrogen, chlorine, and sodium hydroxide production via electrophoretic ion exchange and precipitation.

We demonstrate and analyze a novel desalination method which works by electrophoretically replacing sodium and chloride in feed salt water with a pair of ions, calcium and carbonate, that react and precipitate out. The resulting calcium carbonate precipitate is benign to health, and can be filtered or settled out, yielding low ionic strength product water. The ion exchange and precipitation employs self-sharpening interfaces induced by movement of multiple ions in an electric field to prevent contamination of the product water. Simultaneously, the electrolysis associated with the electromigration produces hydrogen gas, chlorine gas, and sodium hydroxide. We conducted an experimental study of this method's basic efficacy to desalinate salt water from 100 to 600 mol m(-3) sodium chloride. We also present physicochemical models of the process, and analyze replacement reagents consumption, permeate recovery ratio, and energy consumption. We hypothesize that the precipitate can be recycled back to replacement reagents using the well-known, commercially implemented Solvay process. We show that the method's permeate recovery ratio is 58% to 46%, which is on par with that of reverse osmosis. We show that the method's energy consumption requirement over and above that necessary to generate electrolysis is 3 to 10 W h l(-1), which is on par with the energy consumed by state-of-the-art desalination methods. Furthermore, the method operates at ambient temperature and pressure, and uses no specialized membranes. The process may be feasible as a part of a desalination-co-generation facility: generating fresh water, hydrogen and chlorine gas, and sodium hydroxide.

Concentration cascade of leading electrolyte using bidirectional isotachophoresis.

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.

On-chip isotachophoresis for separation of ions and purification of nucleic acids.

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration and efficient separation and extraction based on ionic mobility. For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood. ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species. We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation. We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Electrophoretic mobility measurements of fluorescent dyes using on-chip capillary electrophoresis.

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.

Coupled isotachophoretic preconcentration and electrophoretic separation using bidirectional isotachophoresis.

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.

High-sensitivity detection using isotachophoresis with variable cross-section geometry.

We present a theoretical and experimental study on increasing the sensitivity of ITP assays by varying channel cross-section. We present a simple, unsteady, diffusion-free model for plateau mode ITP in channels with axially varying cross-section. Our model takes into account detailed chemical equilibrium calculations and handles arbitrary variations in channel cross-section. We have validated our model with numerical simulations of a more comprehensive model of ITP. We show that using strongly convergent channels can lead to a large increase in sensitivity and simultaneous reduction in assay time, compared to uniform cross-section channels. We have validated our theoretical predictions with detailed experiments by varying channel geometry and analyte concentrations. We show the effectiveness of using strongly convergent channels by demonstrating indirect fluorescence detection with a sensitivity of 100 nM. We also present simple analytical relations for dependence of zone length and assay time on geometric parameters of strongly convergent channels. Our theoretical analysis and experimental validations provide useful guidelines on optimizing chip geometry for maximum sensitivity under constraints of required assay time, chip area and power supply.

Ionic strength effects on electrophoretic focusing and separations.

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.