Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform.

An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE: This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.

A molecular half-wave rectifier.

This paper describes the performance of junctions based on self-assembled monolayers (SAMs) as the functional element of a half-wave rectifier (a simple circuit that converts, or rectifies, an alternating current (AC) signal to a direct current (DC) signal). Junctions with SAMs of 11-(ferrocenyl)-1-undecanethiol or 11-(biferrocenyl)-1-undecanethiol on ultraflat, template-stripped Ag (Ag(TS)) bottom electrodes, and contacted by top electrodes of eutectic indium-gallium (EGaIn), rectified AC signals, while similar junctions based on SAMs of 1-undecanethiol-SAMs lacking the ferrocenyl terminal group-did not. SAMs in these AC circuits (operating at 50 Hz) remain stable over a larger window of applied bias than in DC circuits. AC measurements, therefore, can investigate charge transport in SAM-based junctions at magnitudes of bias inaccessible to DC measurements. For junctions with SAMs of alkanethiols, combining the results from AC and DC measurements identifies two regimes of bias with different mechanisms of charge transport: (i) low bias (|V| < 1.3 V), at which direct tunneling dominates, and (ii) high bias (|V| > 1.3 V), at which Fowler-Nordheim (FN) tunneling dominates. For junctions with SAMs terminated by Fc moieties, the transition to FN tunneling occurs at |V| ≈ 2.0 V. Furthermore, at sufficient forward bias (V > 0.5 V), hopping makes a significant contribution to charge transport and occurs in series with direct tunneling (V ≲ 2.0 V) until FN tunneling activates (V ≳ 2.0 V). Thus, for Fc-terminated SAMs at forward bias, three regimes are apparent: (i) direct tunneling (V = 0-0.5 V), (ii) hopping plus direct tunneling (V ≈ 0.5-2.0 V), and (iii) FN tunneling (V ≳ 2.0 V). Since hopping does not occur at reverse bias, only two regimes are present over the measured range of reverse bias. This difference in the mechanisms of charge transport at forward and reverse bias for junctions with Fc moieties resulted in large rectification ratios (R > 100) and enabled half-wave rectification.

Cofabrication: a strategy for building multicomponent microsystems.

This Account describes a strategy for fabricating multicomponent microsystems in which the structures of essentially all of the components are formed in a single step of micromolding. This strategy, which we call "cofabrication", is an alternative to multilayer microfabrication, in which multiple layers of components are sequentially aligned ("registered") and deposited on a substrate by photolithography. Cofabrication has several characteristics that make it an especially useful approach for building multicomponent microsystems. It rapidly and inexpensively generates correctly aligned components (for example, wires, heaters, magnetic field generators, optical waveguides, and microfluidic channels) over very large surface areas. By avoiding registration, the technique does not impose on substrates the size limitations of common registrations tools, such as steppers and contact aligners. We have demonstrated multicomponent microsystems with surface areas exceeding 100 cm(2), but in principle, device size is only limited by the requirements of generating the original master. In addition, cofabrication can serve as a low-cost strategy for building microsystems. The technique is amenable to a variety of laboratory settings and uses fabrication tools that are less expensive than those used for multistep microfabrication. Moreover, the process requires only small amounts of solvent and photoresist, a costly chemical required for photolithography; in cofabrication, photoresist is applied and developed only once to produce a master, which is then used to produce multiple copies of molds containing the microfluidic channels. From a broad perspective, cofabrication represents a new processing paradigm in which the exterior (or shell) of the desired structures are produced before the interior (or core). This approach, generating the insulation or packaging structure first and injecting materials that provide function in channels in liquid phase, makes it possible to design and build microsystems with component materials that cannot be easily manipulated conventionally (such as solid materials with low melting points, liquid metals, liquid crystals, fused salts, foams, emulsions, gases, polymers, biomaterials, and fragile organics). Moreover, materials can be altered, removed, or replaced after the manufacturing stage. For example, cofabrication allows one to build devices in which a liquid flows through the device during use, or is replaced after use. Metal wires can be melted and reset by heating (in principle, repairing a break). This method leads to certain kinds of structures, such as integrated metallic wires with large cross-sectional areas or optical waveguides aligned in the same plane as microfluidic channels, that would be difficult or impossible to make with techniques such as sputter deposition or evaporation. This Account outlines the strategy of cofabrication and describes several applications. Specifically, we highlight cofabricated systems that combine microfluidics with (i) electrical wires for microheaters, electromagnets, and organic electrodes, (ii) fluidic optical components, such as optical waveguides, lenses, and light sources, (iii) gels for biological cell cultures, and (iv) droplets for compartmentalized chemical reactions, such as protein crystallization.

Thin, lightweight, foldable thermochromic displays on paper.

This article describes an electronic display that is fabricated by patterning electrically conductive wires (heaters) with micron-scale dimensions on one side of a sheet of paper, and thermochromic ink on the opposite side. Passing electrical current through the wires heats the paper and changes the thermochromic ink from colored (black, green, or other colors) to transparent; this change in property reveals the paper underneath the ink-exposing any messages printed on the paper-and serves as the basis for a two-state "shutter" display. This type of display is thin (100 microm), flat, lightweight (the display weighs <20 mg/cm(2)), can be folded, rolled, twisted, and creased while maintaining function, and ultimately can (if required) be disposed of by incineration. The display is appropriate for applications where information must be presented clearly (usually only once) for little cost (each display costs <$0.10/m(2) in materials) and where limited electrical power is available.

Quantifying colorimetric assays in paper-based microfluidic devices by measuring the transmission of light through paper.

This article describes a point-of-care (POC) system--comprising a microfluidic, paper-based analytical device (micro-PAD) and a hand-held optical colorimeter--for quantifying the concentration of analytes in biological fluids. The micro-PAD runs colorimetric assays, and consists of paper that has been (i) patterned to expose isolated regions of hydrophilic zones and (ii) wet with an index-matching fluid (e.g., vegetable oil) that is applied using a disposable, plastic sleeve encasement. Measuring transmittance through paper represents a new method of quantitative detection that expands the potential functionality of micro-PADs. This prototype transmittance colorimeter is inexpensive, rugged, and fully self-contained, and thus potentially attractive for use in resource-limited environments and developing countries.

Pumping fluids in microfluidic systems using the elastic deformation of poly(dimethylsiloxane).

This paper demonstrates a methodology for storing and pumping fluids that provide a useful capability for microfluidic devices. It uses microfluidic screw valves to isolate fluids in poly(dimethylsiloxane) (PDMS) microcompartments, in which the pressure of the liquid is stored in the elastic deformation of the walls and ceiling of the compartments. Fluids can be stored under pressure in these structures for months. When the valves are opened, the walls and ceiling push the fluid out of the compartments into microfluidic channels. The system has five useful characteristics: (i) it is made using soft lithographic techniques; (ii) it allows multiple reagents to be preloaded in devices and stored under pressure without any additional user intervention; (iii) it makes it possible to meter out fluids in devices, and to control rates of flow of fluids; (iv) it prevents the user from exposure to potentially toxic reagents; and (v) it is hand-operated and does not require additional equipment or resources.

The force acting on a superparamagnetic bead due to an applied magnetic field.

This paper describes a model of the motion of superparamagnetic beads in a microfluidic channel under the influence of a weak magnetic field produced by an electric current passing through a coplanar metal wire. The model based on the conventional expression for the magnetic force experienced by a superparamagnetic bead (suspended in a biologically relevant medium) and the parameters provided by the manufacturer failed to match the experimental data. To fit the data to the model, it was necessary to modify the conventional expression for the force to account for the non-zero initial magnetization of the beads, and to use the initial magnetization and the magnetic susceptibility of the beads as adjustable parameters. The best-fit value of susceptibility deviated significantly from the value provided by the manufacturer, but was in good agreement with the value computed using the magnetization curves measured independently for the beads from the same vial as those used in the experiment. The results of this study will be useful to researchers who need an accurate prediction of the behavior of superparamagnetic beads in aqueous suspensions under the influence of weak magnetic fields. The derivation of the force on a magnetic bead due to a magnetic field also identifies the correct treatment to use for this interaction, and resolves discrepancies present throughout the literature.