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Viral pathogens are a serious health threat around the world, particularly in resource limited settings, where current sensing approaches are often insufficient and slow, compounding the spread and burden of these pathogens. Here, we describe a label-free, point-of-care approach toward detection of virus particles, based on a microfluidic paper-based analytical device with integrated microwire Au electrodes. The device is initially characterized through capturing of streptavidin modified nanoparticles by biotin-modified microwires. An order of magnitude improvement in detection limits is achieved through use of a microfluidic device over a classical static paper-based device, due to enhanced mass transport and capturing of particles on the modified electrodes. Electrochemical impedance spectroscopy detection of West Nile virus particles was carried out using antibody functionalized Au microwires, achieving a detection limit of 10.2 particles in 50 μL of cell culture media. No increase in signal is found on addition of an excess of a nonspecific target (Sindbis). This detection motif is significantly cheaper (∼$1 per test) and faster (∼30 min) than current methods, while achieving the desired selectivity and sensitivity. This sensing motif represents a general platform for trace detection of a wide range of biological pathogens.

Numerous fabrication methods have been reported for microfluidic paper-based analytical devices (μPADs) using barrier materials ranging from photoresist to wax. While these methods have been used with wide success, consistently producing small, high-resolution features using materials and methods that are compatible with solvents and surfactants remains a challenge. Two new methods are presented here for generating μPADs with well-defined, high-resolution structures compatible with solvents and surfactant-containing solutions by partially or fully fusing paper with Parafilm® followed by cutting with a CO laser cutter. Partial fusion leads to laminated paper (-paper) while the complete fusion results in infused paper (-paper). Patterned structures in -paper were fabricated by selective removal of the paper but not the underlying Parafilm® using a benchtop CO laser. Under optimized conditions, a gap as small as 137 ± 22 μm could be generated. Using this approach, a miniaturized paper 384-zone plate, consisting of circular detection elements with a diameter of 1.86 mm, was fabricated in 64 × 43 mm area. Furthermore, these ablation-patterned substrates were confirmed to be compatible with surfactant solutions and common organic solvents (methanol, acetonitrile and dimethylformamide), which has been achieved by very few μPAD patterning techniques. Patterns in -paper were created by completely cutting out zones of the-paper and then fixing pre-cut paper into these openings similar to the strategy of fitting a jigsaw piece into a puzzle. Upon heating, unmodified paper was readily sealed into these openings due to partial reflow of the paraffin into the paper. This unique and simple bonding method was illustrated by two types of 3D μPADs, a push-on valve and a time-gated flow distributor, without adding adhesive layers. The free-standing jigsaw-patterned sheets showed good structural stability and solution compatibility, which provided a facile alternative method for fabricating complicated μPADs.

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Nucleic acid lateral flow assays (NALFAs) have attracted much attention due to their rapid, robust, simple, and cost-effective features. However, the current NALFAs are still limited by low sensitivity because of the poor understanding and control of the underlying complex flow and reaction processes. Although enormous efforts have been devoted to enhancing detection sensitivity of NALFAs, developing simple NALFAs with high sensitivity remains difficult. Thus, we proposed a novel physical−chemical coupling method using dissoluble saline barriers and developed the corresponding mathematical model to better understand the underlying processes to enhance the NALFA sensitivity. Through optimizing the design parameters (e.g., saline barriers patterns, volume, and concentrations) experimentally and numerically, we achieved the highest 10-fold sensitivity enhancement for detection of nucleic acids (including HBV, Staphylococcus aureus, and salmonella as model targets) using this method. The physical−chemical coupling method offers a facile strategy for developing highly sensitive NALFAs.

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