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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]

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]

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]