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This study presents a device and method for multi‐metal printing of microscale structures. The method is based on combining hydrodynamic confinement of electrolytes with localized electrochemical deposition (HLECD), allowing rapid switching of the deposited metal. The device used in HLECD integrates a micro‐anode on a vertical microfluidic chip containing two open‐ended channels that control the flow of electrolytes surrounding the anode. When placed on top of a conducting surface, a localized electrochemical reaction is initiated, wherein the deposited metal composition is dictated by the electrolyte in the confinement. A range of electrolytes, which are used to deposit copper, tin, silver, and nickel, are used to fabricate multiple structures involving more than 60 material changes within a single printing process. The structures are analyzed using electron microscopy, showing the ability to achieve sharp transitions between pure metals. The constant replenishment of electrolytes also eliminates the problem of ion depletion commonly occurring in localized electrochemical deposition, and enables printing rates that are nearly an order of magnitude greater. [ABSTRACT FROM AUTHOR]

Microfluidic paper-based analytical devices (μPADs) rely on capillary flow to achieve filling, mixing and delivery of liquids. We investigate the use of electroosmotic (EO) pumping as a mechanism for dynamic control of capillary flow in paper-based devices. The applied voltage can accelerate or decelerate the baseline capillary-driven velocity, as well as be used to create a tunable valve that reversibly switches the flow on and off in an electrically controlled manner. The method relies on simple fabrication and allows repeated actuation, providing a high degree of flexibility for automation of liquid delivery. We adapt the Lucas–Washburn model to account for EO pumping and provide an experimentally validated analytical model for the distance penetrated by the liquid as a function of time and the applied voltage. We show that the EO-pump can reduce filling time by 6.5-fold for channels spanning several cm in length, relative to capillary filling alone. We demonstrate the utilization of the EO-pump for a tunable and dynamic flow control that accelerates, decelerates and stops the flow on demand. Finally, we present the use of the EO-pump for fluid flow sequencing on a paper-based device. [ABSTRACT FROM AUTHOR]

Microchip electrokinetic methods are capable of increasing the sensitivity of molecular assays by enriching and purifying target analytes. However, their use is currently limited to assays that can be performed under a high external electric field, as spatial separation and focusing is lost when the electric field is removed. We present a novel method that uses two-phase encapsulation to overcome this limitation. The method uses passive filling and pinning of an oil phase in hydrophobic channels to encapsulate electrokinetically separated and focused analytes with a brief pressure pulse. The resulting encapsulated sample droplet maintains its concentration over long periods of time without requiring an electric field and can be manipulated for further analysis, either on- or off-chip. We demonstrate the method by encapsulating DNA oligonucleotides in a 240 pL aqueous segment after isotachophoresis (ITP) focusing, and show that the concentration remains at 60% of the initial value for tens of minutes, a 22-fold increase over free diffusion after 20 minutes. Furthermore, we demonstrate manipulation of a single droplet by selectively encapsulating amplicon after ITP purification from a polymerase chain reaction (PCR) mix, and performing parallel off-chip detection reactions using the droplet. We provide geometrical design guidelines for devices implementing the encapsulation method, and show how the method can be scaled to multiple analyte zones. [ABSTRACT FROM AUTHOR]

We present a novel microfluidic paper-based analytical device (μPAD) which utilizes the native high electroosmotic flow (EOF) in nitrocellulose to achieve stationary isotachophoresis (ITP) focusing. This approach decouples sample accumulation from the length of the channel, resulting in significant focusing over short channel lengths. We provide a brief theory for EOF-balanced ITP focusing under continuous injection from a depleting reservoir and present the design of a short (7 mm) paper-based microfluidic channel, which allows a 200 μL sample to be processed in approximately 6 min, resulting in a 20 000-fold increase in concentration – a full order of magnitude improvement compared to previous paper-based ITP devices. We show the stability of the assay over longer (40 min) durations of time, and using Morpholino probes, we present the applicability of the device for amplification-free detection of nucleic acids, with a limit-of-detection (LoD) of 5 pM in 10 min. Finally, we utilize the small footprint of the channel and show a multiplexed platform in which 12 assays operate in parallel in a 24-well plate format. [ABSTRACT FROM AUTHOR]

Nanostructured porous silicon (PSi) is a promising material for the label-free detection of biomolecules, but it currently suffers from limited applicability due to poor sensitivity, typically in micromolar range. This work presents the design, operation concept, and characterization of a novel microfluidic device and assay that integrates an oxidized PSi optical biosensor with electrokinetic focusing for a highly sensitive label-free detection of nucleic acids. Under proper oxidation conditions, the delicate nanostructure of PSi can be preserved, while providing sufficient dielectric insulation for application of high voltages. This enables the use of signal enhancement techniques, which are based on electric fields. Here, the DNA target molecules are focused using an electric field within a finite and confined zone, and this highly concentrated analyte is delivered to an on-chip PSi Fabry–Pérot optical transducer, pre-functionalized with capture probes. Using reflective interferometric Fourier transform spectroscopy real-time monitoring, a 1000-fold improvement in limit of detection is demonstrated compared to a standard assay, using the same biosensor. Thus, a measured limit of detection of 1 × 10−9 M is achieved without compromising specificity. The concepts presented herein can be readily applied to other ionic targets, paving way for the development of other highly sensitive chemical and biochemical assays. [ABSTRACT FROM AUTHOR]

We present an experimental and analytical study of a novel paper-based analytical device (μPAD) for isotachophoretic sample focusing. Guided by a simple heat transfer model, we further developed wax printing fabrication to enable the creation of shallow channels, which are critical in providing sufficient dissipation of Joule heat, and thus enable the use of high electric fields and short analysis time. This results in a device that is self-contained on a simple piece of filter paper and does not require any specialized enclosures or cooling devices to combat evaporation at high temperatures. Furthermore, we provide an analytical model for isotachophoretic sample accumulation in porous media, introduce a simple figure of merit for evaluating and comparing the efficiency of such devices, and present experimental validation in both paper and glass channels. Using this device we demonstrate the processing of 30 μL of sample achieving 1000-fold increase in peak concentration in 6 min. We believe that this method and device can serve as a guide to the design of low-cost, rapid and highly sensitive paper-based diagnostic platforms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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/m2 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. [Copyright &y& Elsevier]

This paper presents a device ("fluidic biomill") and method for integrated mechanical structuring and biofunctionalization of microfluidic channels, enabling rapid prototyping of biochips. The fluidic biomill leverages hydrodynamic flow confinements (HFCs) to localize the interaction between a fluid and a substrate to a microscale region. By confining organic solvents, and scanning with the biomill over the substrate surface, microchannels as deep as 90 µm are fluidically milled. Subsequently, by switching the confined fluid from a milling solution to a patterning solution, biomolecules are immobilized on the surface of the microchannel. An in‐depth analysis of fluidic milling rates and milled microchannel properties is presented, along with a discussion on the dominant factors in controlling the dimensions of the fluidically milled channel. To demonstrate the utility of this method, a biotin functionalized biochip is fabricated in less than 5 min out of a polystyrene slide and its functionalities are validated by implementing on‐chip electrokinetic sample focusing, pressure driven flow, and a surface‐based reaction. [ABSTRACT FROM AUTHOR]

We leverage electroosmotic-flow generation in porous media in combination with a hydrophobic air gap to create a controllable valve capable of operating in either finite dosing or continuous flow mode, enabling the implementation of multi-step assays on paper-based devices. The hydrophobic air gap between two paper pads creates a barrier keeping the valve nominally closed. Electroosmotic actuation, implemented using a pair of electrodes under the upstream pad, generates sufficient pressure to overcome the barrier and connect the two pads. We present a model describing the flow and governing parameters, including the electric potentials required to open and close the valve and the threshold potential for switching between the modes of operation. We construct the air gap using a hierarchical superhydrophobic surface and study the stability of the closed valve under strenuous conditions and find good agreement between our model and experimental results, as well as stable working conditions for practical applications. We present a straightforward design for a compact and automated device based on paper pads placed on top of printed circuit boards (PCB), equipped with heating and actuation electrodes and additional power and logic capabilities. Finally, we demonstrate the use of the device for amplification of SARS-CoV-2 sequences directly from raw saliva samples, using a loop-mediated isothermal amplification (LAMP) protocol requiring sample lysis followed by enzymatic deactivation and delivery to multiple amplification sites. Since PCB costs scale favorably with mass-production, we believe that this approach could lead to a low-cost diagnostic device that offers the sensitivity of amplification methods. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We present an analytical, numerical, and experimental study of pressure driven counterflow isotachophoresis (ITP). We study solutions to the Nernst-Planck equations in the axi-symmetric and radially dependent case, in the leading order of negligible body forces. We provide a simple model that describes the ITP interface shape for Poiseuille-type counterflows, and an asymptotic model which captures two distinct sample focusing regimes of peak mode ITP. We validate the existence of these regimes using numerical simulations and map the conditions under which each of the focal regions dominates. In particular, we demonstrate numerically that a species diffusivity is a key parameter determining its focusing regime. We experimentally show that this allows spatial separation of co-focusing species having distinctly different diffusivities. We further demonstrate that while dispersion associated with counterflow is typically considered to reduce peak concentrations, certain focusing regimes allow a net gain in sample concentration over the non-dispersed case. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We present an analytical study of peak mode isotachophoresis (ITP), and provide closed form solutions for sample distribution and electric field, as well as for leading-, trailing-, and counter-ion concentration profiles. Importantly, the solution we present is valid not only for the case of fully ionized species, but also for systems of weak electrolytes which better represent real buffer systems and for multivalent analytes such as proteins and DNA. The model reveals two major scales which govern the electric field and buffer distributions, and an additional length scale governing analyte distribution. Using well-controlled experiments, and numerical simulations, we verify and validate the model and highlight its keymerits as well as its limitations. We demonstrate the use of the model for determining the peak concentration of focused sample based on known buffer and analyte properties, and show it differs significantly from commonly used approximations based on the interface width alone. We further apply our model for studying reactions between multiple species having different effective mobilities yet co-focused at a single ITP interface. We find a closed form expression for an effective-on rate which depends on reactants distributions, and derive the conditions for optimizing such reactions. Interestingly, the model reveals that maximum reaction rate is not necessarily obtained when the concentration profiles of the reacting species perfectly overlap. In addition to the exact solutions, we derive throughout several closed form engineering approximations which are based on elementary functions and are simple to implement, yet maintain the interplay between the important scales. Both the exact and approximate solutions provide insight into sample focusing and can be used to design and optimize ITP-based assays. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article presents two approaches for implementation including fluorescence-based and label-free surface profiling, demonstrating the applicability to a variety of sample geometries and surface types. It also leverages point-spread-function engineering for scan-free, dynamic, micro surface profiling.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 fieldeffect 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We present an analytical study of electro-osmotic flow in a Hele-Shaw configuration with non-uniform zeta potential distribution. Applying the lubrication approximation and assuming thin electric double layer, we obtain a pair of uncoupled Poisson equations for the pressure and depth-averaged stream function, and show that the inhomogeneous parts in these equations are governed by gradients in zeta potential parallel and perpendicular to the applied electric field, respectively. We obtain a solution for the case of a disk-shaped region with uniform zeta potential and show that the flow field created is an exact dipole, even in the immediate vicinity of the disk. In addition, we study the inverse problem where the desired flow field is known and solve for the zeta potential distribution required in order to establish it. Finally, we demonstrate that such inverse problem solutions can be used to create directional flows confined within narrow regions, without physical walls. Such solutions are equivalent to flow within channels and we show that these can be assembled to create complex microfluidic networks, composed of intersecting channels and turns, which are basic building blocks in microfluidic devices. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]