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The use of lateral flow assays to detect nucleic acid targets has many applications including point-of-care diagnostics, environmental monitoring, and food safety. A sandwich format, similar to that in protein immunoassays, is often used to capture the target nucleic acid sequence with an immobilized complementary strand anchored to a substrate, and then to visualize this event using a complementary label nucleic acid bound to a nanoparticle label. A critical component of high-sensitivity nucleic acid detection is to utilize high-density capture surfaces for the effective capture of target nucleic acid. Multiple methods have been reported, including the use of streptavidin-based protein anchors that can be adsorbed to the lateral flow substrate and that can utilize the high-affinity streptavidin-biotin linkage to bind biotinylated nucleic acid capture sequences for subsequent target nucleic acid binding. However, these protein anchors have not been systematically characterized for use in the context of nucleic acid detection. In this work, we characterize several protein-based anchors on nitrocellulose for (i) capturing the robustness of the attachment of the protein anchor, (ii) capturing nucleic acid density, and (iii) targeting nucleic acid capture. Further, we demonstrate the signal gains in target nucleic acid hybridization made by increasing the density of capture nucleic acid on a nitrocellulose substrate using multiple applications of protein loading onto nitrocellulose. Finally, we use our high-density capture surfaces to demonstrate high-sensitivity nucleic acid detection in a lateral flow assay (in the context of a SARS-CoV-2 sequence), achieving a LOD of approximately 0.2 nM.

Carbamazepine (CBZ), a drug prescribed to prevent seizures in people with epilepsy, has a narrow therapeutic range such that patients would greatly benefit from personalized drug dosage recommendations. Saliva is an excellent sample for personalized monitoring of CBZ levels because saliva CBZ concentration correlates with the free concentration of CBZ in blood, and can be collected non-invasively. CBZ level quantification using electrochemical detection has been demonstrated in a variety of electrode systems and samples, however, human saliva presents a particular challenge in terms of its complex composition that can result in signal interference via a high background current at the potentials of interest for CBZ detection. Previous demonstrations of electrochemical detection of CBZ in saliva have included rigorous pre-treatment of the sample using centrifugation and high levels of dilution, which is not compatible with lower-resource field settings for patient monitoring of CBZ levels. In this work, we systematically investigate several strategies to improve the detection of CBZ in a background of undiluted human saliva using polymeric laminate-based devices with stencil-printed carbon electrodes; (i) adding the anionic surfactant sodium dodecyl sulfate to the saliva, (ii) filtering saliva to remove larger molecular weight species, (iii) plasma pretreatment of the device electrodes, and (iv) incubation of the sample on the electrodes. These methods enabled the quantification of therapeutically-relevant concentrations of CBZ in a background of human saliva without the need for saliva preprocessing like dilution.

Paper microfluidics is a rapidly growing subfield of microfluidics in which paper-like porous materials are used to create analytical devices that are well-suited for use in field applications. 3D printing technology has the potential to positively affect paper microfluidic device development by enabling tools and methods for the creation of devices with well-defined and tunable fluidic networks of porous matrices for high performance signal generation. This critical review focuses on the progress that has been made in using 3D printing technologies to advance the development of paper microfluidic devices. We describe printing work in three general categories: (i) solid support structures for paper microfluidic device components; (ii) channel barrier definition in existing porous materials; and (iii) porous channels for capillary flow, and discuss their value in advancing paper microfluidic device development. Finally, we discuss major areas of focus for highest impact on the next generation of paper microfluidics devices.

The degradation of biochemical reagents on the timescale of weeks can severely limit the utility of microfluidic assays intended for field use, and is a challenging aspect of microfluidic device development in general. Our study focuses on the evaluation of the dry storage stability of three types of reagents: (i) the colorimetric reagents nitroblue tetrazolium and 1-methoxy-5-methylphenazinium methylsulfate, (ii) the enzyme phenylalanine dehydrogenase, and (iii) the coenzyme β-nicotinamide adenine dinucleotide hydrate, within the context of a phenylalanine monitoring device. We have demonstrated stable dry storage of each of the reagents, over the time span of approximately one month. Drying the colorimetric reagents under nitrogen was found to largely suppress reagent degradation and the appearance of nonspecific signal, while the enzyme and coenzyme retained activity when stored dry for a month without additional processing or chemical additives. Finally, phenylalanine monitoring devices with all three reagent types dried down and stored for 15 days showed comparable functionality to devices containing freshly-dried reagents - a key milestone to enable future clinical testing.

A promising application of paper microfluidics is the translation of gold-standard multi-step laboratory tests to a disposable paper-based format for decentralized diagnostic or therapeutic testing. This often entails conversion of bench-top processing of macro-volume samples to the processing of micro-volume samples within a porous matrix, and requires detailed characterization of fluid and reagent interactions within the porous material(s) of the device. The current study focuses on rational device design through the characterization of fluid and reagent interactions in polysulfone and glass fiber substrates for multi-step sample processing. Specifically, we demonstrate how the characterization of fluidic compatibility between substrates, chemical compatibility between reagents and substrates, sample pH, and sample transport can be used to inform device design in the context of a two-reaction detection scheme for phenylalanine in porous materials. Finally, we demonstrate detection of phenylalanine from human whole blood, and discuss the multiple strengths of the current design over a previous version.