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Paper-based microfluidic devices, also known as microPADs, are an emerging analytical platform with the potential to improve point-of-care diagnostics. MicroPADs are fabricated by patterning hydrophobic inks onto sheets of paper to create hydrophilic channels and test zones. One of the main advantages of microPADs is that they are inexpensive and simple to fabricate, making them accessible even to researchers with limited budgets or no prior fabrication expertise. Wax printing, where a solid ink printer is used to pattern wax on paper, has been the most convenient and popular method for fabricating paper-based microfluidic devices. Unfortunately, solid ink printers were discontinued in 2016 and are no longer available commercially. Here we introduce a method for fabricating microPADs using a portable thermal transfer printer that retains the convenience of wax printing. Devices fabricated by thermal transfer printing were comparable to devices fabricated via wax printing and laser printing. The low cost, convenience, and portability of the thermal transfer printer make this approach an exciting prospect for replacing wax printing and facilitating the continued development of paper-based microfluidics.

Shrinking paper money is a captivating and memorable demonstration of chemistry in action that can be carried out using minimal equipment and reagents. Paper bills can be shrunk down to ∼25% of their initial surface area by treating them with an aqueous solution of sodium periodate. The cellulose in the paper bill is oxidized to dialdehyde cellulose via the Malaprade reaction, and the resulting change in size of the paper money provides macroscopic evidence of the reaction. The chemical change that occurs in the cellulose can be confirmed by a Tollen’s test.

Three commercially-available conductive filaments were evaluated for 3D printing flexible circuits on paper. While all three filaments were printed successfully, the resulting conductive traces were found to have significantly different impedances when characterized by electrochemical impedance spectroscopy. Using a graphite-doped polylactic acid filament, the flexibility of paper-based conductive traces was evaluated, methods of integrating common electrical and electronic components with the conductive traces were demonstrated, and the resistive heating of the traces was characterized. The ability to 3D print conductive traces on paper using commercially available materials opens many opportunities for rapid prototyping of flexible electronics and for integrating electronic circuits with paper-based microfluidic devices.

Cellulose-based paper is a versatile material with a diverse array of applications. While paper is not commonly thought of as a material that shrinks, here we present a method for miniaturizing paper via periodate oxidation. Chromatography paper was exposed to varying concentrations of periodate (0.1–0.5 M) over a 96-h period. Following optimization of miniaturization parameters, fourteen different types of paper were miniaturized and reductions in surface area ranging from 60 to 80% were observed. All cellulose paper types, but not cellulose-derivatives, displayed successful miniaturization. Results were highly tunable dependent upon periodate concentration and reaction time. Potential applications of the technique are discussed, including its use as a microfabrication method.

The following article summarizes United States Patent Application No. US20180052155A1, titled ‘Assay Devices and Methods’ (filed 16 August 2016, published 22 February 2018). While lateral flow assays (LFAs) have revolutionized point-of-care diagnostics by enabling accurate, inexpensive, and rapid detection of biomarkers, they typically do not provide quantitative results. Hence, there is a significant need for quantitative assays at the point of care. This patent summary describes a novel method of chronometric biomarker quantitation via enzymatic degradation of a metastable gelatin-based biomatrix, principally suited for use in paper-based microfluidic devices (microPADs). This new quantitation mechanism was designed to meet the ASSURED criteria for point-of-care diagnostic devices laid forth by the World Health Organization and may ultimately provide increased access to healthcare, at a significantly reduced cost, around the world.

Microbial staining techniques are widely employed in clinical and academic laboratories for classifying and identifying microorganisms derived from clinical, food and environmental samples. Staining allows for the rapid visualization and determination of many morphological characteristics of microorganisms, used for their identification and classification. Over the past century, staining techniques such as the Gram stain, the Capsule stain, the Acid-fast stain and the Endospore stain, have seen few advances, and manual staining remains the gold standard. Typical instructions for these staining procedures recommend 'flooding' glass slides with milliliter volumes of dye, resulting in large volumes of hazardous waste. Here we present micro-staining, a simple alternative to flooding that utilizes microliter volumes of dye. Micro-staining minimizes the volume of waste generated, leads to significant cost savings for the laboratory, requires limited training, and produces results with equivalent quality to traditional stains.

Microfluidic paper-based analytical devices (microPADs) have emerged as a promising platform for point-of-care diagnostic devices. While the inherent wicking properties of microPADs allow for fluid flow without supporting equipment, this also presents a major challenge in achieving robust fluid control, which becomes especially important when performing complex multi-step assays. Herein, we describe an ideal method of fluid control mediated by wax-printed fluidic time delays. This method relies on a simple fabrication technique, does not utilize chemicals/reagents that could affect downstream assays, is readily scalable, and has a wide temporal range of tunable fluid control. The delays are wax printed on both the top and bottom of pre-fabricated microPAD channels, without subsequent heating, to create hemi-/fully-enclosed channels. With these wax printed delays, we were able to tune the time it took aqueous solutions to wick across a 25 mm-long channel between 3.6 min and 13.4 min. We then employed these fluid delays in the sequential delivery of four dyes to a test zone. Additionally, we demonstrated the automation of two simple enzymatic assays with this fluid control modality. This method of fluid control may allow future researchers to automate more complex assays, thereby further advancing microPADs toward real-world applications.

Microfluidic paper-based analytical devices (microPADs) are emerging as cost-effective and portable platforms for point-of-care assays. A fundamental limitation of microPAD fabrication is the imprecise nature of most methods for patterning paper. The present work demonstrates that paper patterned via wax printing can be miniaturized by treating it with periodate to produce higher-resolution, high-fidelity microPADs. The optimal miniaturization parameters were determined by immersing microPADs in various concentrations of aqueous sodium periodate (NaIO4) for varying lengths of time. This treatment miniaturized microPADs by up to 80% in surface area, depending on the concentration of periodate and length of the reaction time. By immersing microPADs in 0.5-M NaIO4 for 48 hours, devices were miniaturized by 78% in surface area, and this treatment allowed for the fabrication of functional channels with widths as small as 301 µm and hydrophobic barriers with widths as small as 387 µm. The miniaturized devices were shown to be compatible with redox-based colorimetric assays and enzymatic reactions. This miniaturization technique provides a new option for fabricating sub-millimeter-sized features in paper-based fluidic devices without requiring specialized equipment and could enable new capabilities and applications for microPADs.

Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices. The technique is as simple as drawing with the reagent pencils on a device. When aqueous samples are added to the device, the reagents dissolve from the pencil matrix and become available to react with analytes in the sample. Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution. Most importantly, sensitive reagents, such as enzymes, are stable in the pencils under ambient conditions, and no significant decrease in the activity of the enzyme horseradish peroxidase stored in a pencil was observed after 63 days. Reagent pencils offer a new option for preparing and customizing diagnostic tests at the point of care without the need for specialized equipment.

Paper microzone plates in combination with a noncontact liquid handling robot were demonstrated as tools for studying the stability of enzymes stored on paper. The effect of trehalose and SU-8 epoxy novolac resin (SU-8) on the stability of horseradish peroxidase (HRP) was studied in both a short-term experiment, where the activity of various concentrations of HRP dried on paper were measured after 1 h, and a long-term experiment, where the activity of a single concentration of HRP dried and stored on paper was monitored for 61 days. SU-8 was found to stabilize HRP up to 35 times more than trehalose in the short-term experiment for comparable concentrations of the two reagents, and a 1% SU-8 solution was found to stabilize HRP approximately 2 times more than a 34% trehalose solution in both short- and long-term experiments. The results suggest that SU-8 is a promising candidate for use as an enzyme-stabilizing reagent for paper-based diagnostic devices and that the short-term experiment could be used to quickly evaluate the capacity of various reagents for stabilizing enzymes to identify and characterize new enzyme-stabilizing reagents.

Retroviral Gag proteins are synthesized as soluble, myristoylated precursors that traffic to the plasma membrane and promote viral particle production. The intracellular transport of human immunodeficiency virus type 1 (HIV-1) Gag to the plasma membrane remains poorly understood, and cellular motor proteins responsible for Gag movement are not known. Here we show that disrupting the function of KIF4, a kinesin family member, slowed temporal progression of Gag through its trafficking intermediates and inhibited virus-like particle production. Knockdown of KIF4 also led to increased Gag degradation, resulting in reduced intracellular Gag protein levels; this phenotype was rescued by reintroduction of KIF4. When KIF4 function was blocked, Gag transiently accumulated in discrete, perinuclear, nonendocytic clusters that colocalized with endogenous KIF4, with Ubc9, an E2 SUMO-1 conjugating enzyme, and with SUMO. These studies identify a novel transit station through which Gag traffics en route to particle assembly and highlight the importance of KIF4 in regulating HIV-1 Gag trafficking and stability.

Bacillus anthracis lethal toxin (LT) produces symptoms of anthrax in mice and induces rapid lysis of macrophages (Mφ) derived from certain inbred strains. We used nine inbred strains and two inducible nitric oxide synthase (iNOS) knockout C57BL/6J strains polymorphic for the LT Mφ sensitivity Kif1C locus to analyze the role of Mφ sensitivity (to lysis) in LT-mediated cytokine responses and lethality. LT-mediated induction of cytokines KC, MCP-1/JE, MIP-2, eotaxin, and interleukin-1β occurred only in mice having LT-sensitive Mφ. However, while iNOS knockout C57BL/6J mice having LT-sensitive Mφ were much more susceptible to LT than the knockout mice with LT-resistant Mφ, a comparison of susceptibilities to LT in the larger set of inbred mouse strains showed a lack of correlation between Mφ sensitivity and animal susceptibility to toxin. For example, C3H/HeJ mice, harboring LT-sensitive Mφ and having the associated LT-mediated cytokine response, were more resistant than mice with LT-resistant Mφ and no cytokine burst. Toll-like receptor 4 (Tlr4)-deficient, lipopolysaccharide-nonresponsive mice were not more resistant to LT. We also found that CAST/Ei mice are uniquely sensitive to LT and may provide an economical bioassay for toxin-directed therapeutics. The data indicate that while the cytokine response to LT in mice requires Mφ lysis and while Mφ sensitivity in the C57BL/6J background is sufficient for BALB/cJ-like mortality of that strain, the contribution of Mφ sensitivity and cytokine response to animal susceptibility to LT differs among other inbred strains. Thus, LT-mediated lethality in mice is influenced by genetic factors in addition to those controlling Mφ lysis and cytokine response and is independent of Tlr4 function.