Moran Bercovici

Open source simulation tool for electrophoretic stacking, focusing, and separation.

We present the development, formulation, and performance of a new simulation tool for electrophoretic preconcentration and separation processes such as capillary electrophoresis, isotachophoresis, and field amplified sample stacking. The code solves the one-dimensional transient advection-diffusion equations for multiple multivalent weak electrolytes (including ampholytes) and includes a model for pressure-driven flow and Taylor-Aris dispersion. The code uses a new approach for the discretization of the equations, consisting of a high resolution compact scheme which is combined with an adaptive grid algorithm. We show that this combination allows for accurate resolution of sharp concentration gradients at high electric fields, while at the same time significantly reducing the computational time. We demonstrate smooth, stable, and accurate solutions at current densities as high as 5000A/m(2) using only 300 grid points, and a 75-fold reduction in computational time compared with equivalent uniform grid techniques. The code is available as an open source for free at http://microfluidics.stanford.edu.

Rapid detection of urinary tract infections using isotachophoresis and molecular beacons

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.

Rapid hybridization of nucleic acids using isotachophoresis

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.

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.

Clinical Validation of Integrated Nucleic Acid and Protein Detection on an Electrochemical Biosensor Array for Urinary Tract Infection Diagnosis

Background Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management. Methodology/Principal Findings The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both). Conclusion/Significance We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.

Acceleration of surface-based hybridization reactions using isotachophoretic focusing.

We present a theoretical model and experimental demonstration of a novel method for acceleration of surface-based reactions using isotachophoresis (ITP). We use ITP to focus a sample of interest and deliver a high concentration target to a prefunctionalized surface, thus enabling rapid reaction at the sensor site. The concentration of the focused analyte is bound in space by the ITP interface and, upon reaction with the surface, continues electromigrating downstream, removing any contamination or reacted sample molecules from the surface. This constitutes a one-step react-and-wash assay which can be performed in a simple channel and does not require flow control elements or moving parts. We designed a novel microfluidic chip where reaction surfaces are formed by paramagnetic beads, immobilized at desired sites by an external magnetic field. Using this chip, we compared ITP-based surface hybridization to standard continuous flow-based hybridization and experimentally demonstrated a 2 orders of magnitude improvement in limit of detection (LoD) in a 3 min nucleic acid hybridization assay. The simple analytical model we present allows prediction of the rate of surface reaction under ITP and can be used to design and optimize such assays as a function of the physical properties of the system, including buffer chemistry, applied voltage, analyte mobility, analyte concentration, probe density, and surface length. The method, model, and experimental setup can be applied to various forms or surface reactions and may serve as the basis for highly genetic analysis and immunoassays.

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.

Microfluidic assay for continuous bacteria detection using antimicrobial peptides and isotachophoresis.

We present a novel microfluidic assay for continuous and quantitative detection of bacteria in water. We leverage isotachophoresis (ITP), an electrophoretic focusing technique, to create a stationary high concentration zone of fluorescently labeled antimicrobial peptides (AMPs) in a microfluidic channel. The tested water sample flows continuously through this high concentration AMPs reaction zone; any bacteria present in the sample is simultaneously labeled by, and separated from, the high concentration AMPs. The labeled bacteria continue into the downstream pure-buffer zone where the fluorescence signal is monitored, providing a direct quantitative measurement of the original bacterial concentration in the sample. We present the principles of the technique, demonstrate its applicability for quantitative detection of E. coli as well as its stability over a 1 h monitoring time, and provide a simple model for predicting its performance at different operating conditions. The method could be potentially expanded for use with other types of probes and provide continuous analysis and monitoring of water samples at the point of need.

Compact adaptive-grid scheme for high numerical resolution simulations of isotachophoresis.

In a previous publication we demonstrated a fast simulation tool for solution of electrophoretic focusing and separation. We here describe the novel mathematical model and numerical algorithms used to create this code. These include the representation of advection-diffusion equations on an adaptive grid, high-resolution discretization of the equations (sixth order compact), a new variational-based approach for controlling the motion of grid points, and new boundary conditions which enable solution in a moving frame of reference. We discuss the advantages of combining a high-resolution discretization with an adaptive grid in accurately resolving sharp interfaces in isotachophoresis, and provide verification against known analytical solutions and comparison with prevailing exiting numerical algorithms.

Fluorescent Carrier Ampholytes Assay for Portable, Label-Free Detection of Chemical Toxins in Tap Water

We present a novel method for fluorescence-based indirect detection of analytes and demonstrate its use for label-free detection of chemical toxins in a hand-held device. We fluorescently label a mixture of low-concentration carrier ampholytes and introduce it into an isotachophoresis (ITP) separation. The carrier ampholytes provide a large number of fluorescent species with a wide range of closely spaced effective electrophoretic mobilities. Analytes focus under ITP and displace subsets of these carrier ampholytes. The analytes are detected indirectly and quantified by analyzing the gaps in the fluorescent ampholyte signal. The large number (on the order of 1000) of carrier ampholytes enables detection of a wide range of analytes, requiring little a priori knowledge of their electrophoretic properties. We discuss the principles of the technique and demonstrate its use in the detection of various analytes using a standard microscope system. We then present the integration of the technique into a self-contained hand-held device and demonstrate detection of chemical toxins (2-nitrophenol and 2,4,6-trichlorophenol) in tap water, with no sample preparation steps.