Selective Extraction of Biomolecules Using a Bidirectional Flow Filter.

We present a microfluidic device for selective separation and extraction of molecules based on their diffusivity. The separation relies on electroosmotically driven bidirectional flows in which high-diffusivity species experience a net-zero velocity and lower diffusivity species are advected to a collection reservoir. The device can operate continuously and is suitable for processing low sample volumes. Using several model systems, we show that the extraction efficiency of the system is maintained at more than 90% over tens of minutes with a purity of more than 99%. We demonstrate the applicability of the device to the extraction of genomic DNA from short DNA fragments.

Dynamic microscale flow patterning using electrical modulation of zeta potential.

The ability to move fluids at the microscale is at the core of many scientific and technological advancements. Despite its importance, microscale flow control remains highly limited by the use of discrete channels and mechanical valves, and relies on fixed geometries. Here we present an alternative mechanism that leverages localized field-effect electroosmosis to create dynamic flow patterns, allowing fluid manipulation without the use of physical walls. We control a set of gate electrodes embedded in the floor of a fluidic chamber using an ac voltage in sync with an external electric field, creating nonuniform electroosmotic flow distributions. These give rise to a pressure field that drives the flow throughout the chamber. We demonstrate a range of unique flow patterns that can be achieved, including regions of recirculating flow surrounded by quiescent fluid and volumes of complete stagnation within a moving fluid. We also demonstrate the interaction of multiple gate electrodes with an externally generated flow field, allowing spatial modulation of streamlines in real time. Furthermore, we provide a characterization of the system in terms of time response and dielectric breakdown, as well as engineering guidelines for its robust design and operation. We believe that the ability to create tailored microscale flow using solid-state actuation will open the door to entirely new on-chip functionalities.

Microscale Hydrodynamic Cloaking and Shielding via Electro-Osmosis.

We demonstrate theoretically and experimentally that injection of momentum in a region surrounding an object in microscale flow can yield both "cloaking" conditions, where the flow field outside the cloaking region is unaffected by the object, and "shielding" conditions, where the hydrodynamic forces on the object are eliminated. Using field-effect electro-osmosis as a mechanism for injection of momentum, we present a theoretical framework and analytical solutions for a range of geometrical shapes, validate these both numerically and experimentally, and demonstrate the ability to dynamically switch between the different states.

Electrokinetic Scanning Probe.

The theoretical analysis and experimental demonstration of a new concept are presented for a non-contact scanning probe, in which transport of fluid and molecules is controlled by electric fields. The electrokinetic scanning probe (ESP) enables local chemical and biochemical interactions with surfaces in liquid environments. The physical mechanism and design criteria for such a probe are presented, and its compatibility with a wide range of processing solutions and pH values are demonstrated. The applicability of the probe is shown for surface patterning in conjunction with localized heating and 250-fold analyte stacking.

Nonuniform Electro-osmotic Flow Drives Fluid-Structure Instability.

We demonstrate the existence of a fluid-structure instability arising from the interaction of electro-osmotic flow with an elastic substrate. Considering the case of flow within a soft fluidic chamber, we show that above a certain electric field threshold, negative gauge pressure induced by electro-osmotic flow causes the collapse of its elastic walls. We combine experiments and theoretical analysis to elucidate the underlying mechanism for instability and identify several distinct dynamic regimes. The understanding of this instability is important for the design of electrokinetic systems containing soft elements.

Intermediate States of Wetting on Hierarchical Superhydrophobic Surfaces.

Wetting transition on superhydrophobic surfaces is commonly described as an abrupt jump between two stable states-either from Cassie to Wenzel for nonhierarchical surfaces or from Cassie to nano-Cassie on hierarchical surfaces. We here experimentally study the electrowetting of hierarchical superhydrophobic surfaces composed of multiple length scales by imaging the light reflections from the gas-liquid interface. We present the existence of a continuous set of intermediate states of wetting through which the gas-liquid interface transitions under a continuously increasing external forcing. This transition is partially reversible and is limited only by localized Cassie to Wenzel transitions at nanodefects in the structure. In addition, we show that even a surface containing many localized wetted regions can still exhibit extremely low contact angle hysteresis, thus remaining useful for many heat transfer and self-cleaning applications. Expanding the classical definition of the Cassie state in the context of hierarchical surfaces, from a single state to a continuum of metastable states ranging from the centimeter to the nanometer scale, is important for a better description of the slip properties of superhydrophobic surfaces and provides new considerations for their effective design.

Rapid phenotypic antimicrobial susceptibility testing using nanoliter arrays.

Antibiotic resistance is a major global health concern that requires action across all sectors of society. In particular, to allow conservative and effective use of antibiotics clinical settings require better diagnostic tools that provide rapid determination of antimicrobial susceptibility. We present a method for rapid and scalable antimicrobial susceptibility testing using stationary nanoliter droplet arrays that is capable of delivering results in approximately half the time of conventional methods, allowing its results to be used the same working day. In addition, we present an algorithm for automated data analysis and a multiplexing system promoting practicality and translatability for clinical settings. We test the efficacy of our approach on numerous clinical isolates and demonstrate a 2-d reduction in diagnostic time when testing bacteria isolated directly from urine samples.

Tunable Bidirectional Electroosmotic Flow for Diffusion-Based Separations.

We present a new concept for on-chip separation that leverages bidirectional flow, to tune the dispersion regime of molecules and particles. The system can be configured so that low diffusivity species experience a ballistic transport regime and are advected through the chamber, whereas high diffusivity species experience a diffusion dominated regime with zero average velocity and are retained in the chamber. We detail the means of achieving bidirectional electroosmotic flow using an array of alternating current (AC) field-effect electrodes, experimentally demonstrate the separation of particles and antibodies from dyes, and present a theoretical analysis of the system, providing engineering guidelines for its design and operation.

Electroosmotic Flow Dipole: Experimental Observation and Flow Field Patterning.

We experimentally demonstrate the phenomenon of electroosmotic dipole flow that occurs around a localized surface charge region under the application of an external electric field in a Hele-Shaw cell. We use localized deposition of polyelectrolytes to create well-controlled surface charge variations, and show that, for a disk-shaped spot, the internal pressure distribution that arises results in uniform flow within the spot and dipole flow around it. We further demonstrate the superposition of surface charge spots to create complex flow patterns, without the use of physical walls.

Spatially Resolved Genetic Analysis of Tissue Sections Enabled by Microscale Flow Confinement Retrieval and Isotachophoretic Purification.

We have developed a method for spatially resolved genetic analysis of formalin-fixed paraffin-embedded (FFPE) cell block and tissue sections. This method involves local sampling using hydrodynamic flow confinement of a lysis buffer, followed by electrokinetic purification of nucleic acids from the sampled lysate. We characterized the method by locally sampling an array of points with a circa 200 µm diameter footprint, enabling the detection of single KRAS and BRAF point mutations in small populations of RKO and MCF-7 FFPE cell blocks. To illustrate the utility of this approach for genetic analysis, we demonstrate spatially resolved genotyping of FFPE sections of human breast invasive ductal carcinoma.

Real-Time Monitoring of Fluorescence in Situ Hybridization Kinetics.

We present a novel method for real-time monitoring and kinetic analysis of fluorescence in situ hybridization (FISH). We implement the method using a vertical microfluidic probe containing a microstructure designed for rapid switching between probe solution and nonfluorescent imaging buffer. The FISH signal is monitored in real time during the imaging buffer wash, during which signal associated with unbound probes is removed. We provide a theoretical description of the method as well as a demonstration of its applicability using a model system of centromeric probes (Cen17). We demonstrate the applicability of the method for characterization of FISH kinetics under conditions of varying probe concentration, destabilizing agent (formamide) content, volume exclusion agent (dextran sulfate) content, and ionic strength. We show that our method can be used to investigate the effect of each of these variables and provide insight into processes affecting in situ hybridization, facilitating the design of new assays.

Nanoliter Cell Culture Array with Tunable Chemical Gradients.

A multitude of cell screening assays for diagnostic and research applications rely on quantitative measurements of a sample in the presence of different reagent concentrations. Standard methods rely on microtiter plates of varying well density, which provide simple and standardized sample addressability. However, testing hundreds of chemical dilutions requires complex automation, and typical well volumes of microtiter plates are incompatible with the analysis of a small number of cells. Here, we present a microfluidic device for creating a high-resolution chemical gradient spanning 200 nanoliter wells. Using air-based shearing, we show that the individual wells can be compartmentalized without altering the concentration gradient, resulting in a large set of isolated nanoliter cell culture wells. We provide an analytical and numerical model for predicting the concentration within each culture chamber and validate it against experimental results. We apply our system for the investigation of yeast cell metabolic gene regulation in the presence of different ratios of galactose/glucose concentrations and successfully resolve the nutrient threshold at which the cells activate the galactose pathway.

Monitoring Dissociation Kinetics during Electrophoretic Focusing to Enable High-Specificity Nucleic Acid Detection.

A wide range of medical conditions can be diagnosed through sequence-specific analysis of nucleic acids. However, a major challenge remains in detecting a specific target in samples containing a high concentration of mismatching sequences. A single-step kinetic homogenous (free solution) assay is presented in which free sequence-specific probes are continuously separated from probe-target hybrids during electrophoretic sample focusing, allowing monitoring of dissociation kinetics. Under these conditions, the different kinetics of targets versus mismatches result in distinct patterns of the signal (for example, linear increase for target versus exponential decay for mismatch), allowing the detection of desired sequences even in the presence of high background nucleic acid content. Additionally, an analytical model provides insight into the underlying dynamics, and allows design of assays based on this mechanism.

Isotachophoresis-Based Surface Immunoassay.

In the absence of amplification methods for proteins, the immune-detection of low-abundance proteins using antibodies is fundamentally limited by binding kinetic rates. Here, we present a new class of surface-based immunoassays in which protein-antibody reaction is accelerated by isotachophoresis (ITP). We demonstrate the use of ITP to preconcentrate and deliver target proteins to a surface decorated with specific antibodies, where effective utilization of the focused sample is achieved by modulating the driving electric field (stop-and-diffuse ITP mode) or applying a counter flow that opposes the ITP motion (counterflow ITP mode). Using enhanced green fluorescent protein (EGFP) as a model protein, we carry out an experimental optimization of the ITP-based immunoassay and demonstrate a 1300-fold improvement in limit of detection compared to a standard immunoassay, in a 6 min protein-antibody reaction. We discuss the design of buffer chemistries for other protein systems and, in concert with experiments, provide full analytical solutions for the two operation modes, elucidating the interplay between reaction, diffusion, and accumulation time scales and enabling the prediction and design of future immunoassays.

Focused upon hybridization: rapid and high sensitivity detection of DNA using isotachophoresis and peptide nucleic acid probes.

We present a novel assay for rapid and high sensitivity detection of nucleic acids without amplification. Utilizing the neutral backbone of peptide nucleic acids (PNA), our method is based on the design of low electrophoretic mobility PNA probes, which do not focus under isotachophoresis (ITP) unless bound to their target sequence. Thus, background noise associated with free probes is entirely eliminated, significantly improving the signal-to-noise ratio while maintaining a simple single-step assay requiring no amplification steps. We provide a detailed analytical model and experimentally demonstrate the ability to detect targets as short as 17 nucleotides (nt) and a limit of detection of 100 fM with a dynamic range of 5 decades. We also demonstrate that the assay can be successfully implemented for detection of DNA in human serum without loss of signal. The assay requires 15 min to complete, and it could potentially be used in applications where rapid and highly sensitive amplification-free detection of nucleic acids is desired.

Current monitoring in a microchannel with repeated constrictions for accurate detection of sample location in isotachophoresis.

We present a new method for accurate detection of sample location in peak mode isotachophoresis (ITP). The technique is based on the design of a microchannel with multiple constrictions and on detecting the passage of the ITP interface through these constrictions. We achieve this by monitoring the electric current across the channel, which exhibits sharp decreases as the ITP interface moves more rapidly through the higher current density constrictions. We show that cross-correlation between the electric current signal and a predefined step function is an effective method for detecting changes in the slope of the electric current curve in real-time and is robust to changes in the composition of the buffers. We demonstrate the use of the technique to deliver sample to a designated location in the channel with an accuracy as low as 50 µm. Importantly, the method does not require the use of any optics and thus can be used to monitor the location of unlabeled species for application in a variety of ITP assays.

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.

Simulation tool coupling nonlinear electrophoresis and reaction kinetics for design and optimization of biosensors.

We present the development, formulation, validation, and demonstration of a fast, generic, and open source simulation tool, which integrates nonlinear electromigration with multispecies nonequilibrium kinetic reactions. The code is particularly useful for the design and optimization of new electrophoresis-based bioanlaytical assays, in which electrophoretic transport, separation, or focusing control analyte spatial concentration and subsequent reactions. By decoupling the kinetics solver from the electric field solver, we demonstrate an order of magnitude improvement in total simulation time for a series of 100 reaction simulations using a shared background electric field. The code can efficiently handle complex electrophoretic setups coupling sharp electric field gradients with bulk reactions, surface reactions, and competing reactions. For example, we demonstrate the use of the code for investigating accelerated reactions using isotachophoresis (ITP), revealing new regimes of operation which in turn enable significant improvement of the signal-to-noise ratio of ITP-based genotypic assays. The user can define arbitrary initial conditions and reaction rules, and we believe it will be a valuable tool for the design of novel bioanalytical assays. We will offer the code as open source, and it will be available for free download at http://microfluidics.technion.ac.il.

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.